Plastome Analysis and Phylogenetic Reconstruction of Iris Speci es in China

Plastome Analysis and Phylogenetic Reconstruction of Iris Speci es in China | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Plastome Analysis and Phylogenetic Reconstruction of Iris Speci es in China jinfeng liu, Xin Jin, Xingtang Du, yanping xie, xianfeng Jiang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7909828/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 19 You are reading this latest preprint version Abstract Background Iris is a group of perennial herb with important horticultural value. There are over 300 species of Iris species worldwide, and China has approximately 70 species. A series of studies utilized chloroplast fragments to explore the phylogenetic relationships of Irises, but the number of species and the reliability of the results of most studies were not satisfied. We sequenced and assembled the chloroplast genomes of 17 species of Iris , and downloaded 62 available data from public database to construct the most complete phylogenetic evolutionary tree and analyze the evolution and origin relationship of genus Iris. Results The 79 Iris chloroplast genomes exhibit highly similar genome size, gene content, and order. The Iris chloroplast genomes show typical quadripartite structures with lengths from 150,169 bp to 155,878 bp. All plastomes exhibit typical quadripartite structure, consisting of a large single-copy (LSC) region, a small single-copy (SSC) region, and two inverted repeats (IRa and IRb). Phylogenetic results support Iris as a monophyletic group, and indicate that Iris was divided into three major clades. The divergence times indicate that Iris diverged from Crocos at early Eocenein. Bearded and rhizomelic might be the original traits of Iris , and then independently evolve into beardless, bulbous or tuberous. Conclusion Our study supports the present taxonomic treatment at the subgenus level for Iris species, and demonstrates the validity of phylogenetic resolution using whole chloroplast genome sequences. We also prove that the Iris plastome developed molecular markers can help us better identify and understand the evolutionary history of Iris species in the future. Epilepsy Stigma Depression Quality of life Rural Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Irises are a group of perennial herbaceous plants widely distributed across temperate regions of Eurasia [ 1 ]. There are around 300 Iris species globally, with Europe to Central Asia being one of the major diversity center in the world. Iris is highly valued for its striking beauty and economic uses [ 2 – 5 ]. As a group of well-known horticultural plants, it has a very long cultivation history across most civilization of the world [ 1 ]. The cultivation of Irises could date back to ancient Egypt, Asia Minor, and ancient India as early as 3000 BCE [ 1 ]. In ancient Greece, Iris is regarded as the embodiment of the divine. The name of the genus, Iris , comes from the Greek goddess of the rainbow, who served as a messenger between gods and mortals [ 1 ]. China is home to nearly 70 Iris species, accounting for nearly one-fifth of world's Irises species, primarily distributed across southwest, northwest, and northeast China [ 6 ]. Until now, a series of newly described Iris species continue to be found and documented in China [ 7 – 9 ]. In Flora of China, Irises in China were assigned into six subgenera and 7 sections based on the geographical distribution, morphological characteristics and genetic relationships [ 6 ], largely follows Rodionenko's system [ 10 ]. Irises produce conspicuous flowers that are characterized by colourful perianth whorls, drooping lower petals (falls), elevated upper petals (standards), nectary guides (beard, crest or stain), and petaloid style branches [ 11 ]. Generally speaking, the flowers of wild Irises are usually blue, yellow or red [ 12 ]. The basal leaves of Iris form a fan-shaped rosette, with a few reduced cauline leaves emerging from the reproductive stem. Iris species exhibit a variety of underground organs, including rhizomes, bulbs, tuberous roots, and stolons. The crest on the falls of Iris displays high diversity that may be bearded or beardless [ 13 ]. Taxonomically, Iris is divided into numerous subgenera primarily based on the traits of underground organs, crest on falls, and seed arils [ 13 , 14 ]. The crests on falls play crucial role in the taxonomic subgenera classification of Iris . The first comprehensive systematic study of Iris [ 3 ] categorized approximately 140 species into 12 sections, with crested species placed in three sections. Six crested species with stout rhizomes were referred to sect. Evansia , and the crested species with fleshy rootstocks were assigned to sect. Nepalensis . Lawrence [ 13 ] downgraded sect. Evansia to subsection and raised sect. Nepalensis to subg. Nepalensis . In 1987, Rodionenko [ 10 ] promoted subsect. Evansia to subg. Crossiris . Mathew [ 15 ] adopted Lawrence’s [ 13 ] taxonomic definitions and elevated the subsection Evansia to sectional level, placing it within subg. Limniris , as sect. Lophiris . Underground organ is one of the other important classification criterions in Iris . Dykes [ 3 ] proposed that rhizomes and bulbs are the original underground traits of Iris . These two traits firstly appeared in subgenus Limniris , and then evolve to other species of Iris . Taking Iris grant-duffii as an example (a species which possesses bulbous root and rhizomes, simultaneously), he also proposed that the bulbous root was more original, and subsequently replaced by rhizomes. Lawrence [ 13 ] classified the bulbous species of Iris into two subgenera, the subgenus Xiphium has bulbous roots but lacking of fleshy roots, and subgenus Scorpiris has both bulbous roots and fleshy roots. The genus Iris has long been recognized as monophyletic group. However, various phylogeny study indicated that whether it’s a monophyly still remain controversial [ 11 , 16 , 17 ]. For example, Wilson [ 14 ] reconstructed the cladogram of Iris based on three chloroplast DNA fragments for 104 Iris species indicated that three monotypic genera Belamcanda, Pardanthopsis, and Hermodactylus clustered within the same clade with Iris . Furthermore, the inter-specific relationships within Iris are much more complex [ 12 , 18 ]. For examples, except for the subgen. Nepalensis and the subgen. Xiphium , all the subgenera of Iris were identified as non-monophyletic [ 14 , 16 ]. As chloroplast genome could offer significantly more abundant information compared to traditional PCR-amplified fragments [ 19 , 20 ]. This study will collect and sequence the chloroplast genomes of 79 Irises to investigate the genomic characteristics and evolutionary relationship within this group. Materials and methods 1.1 Data and plant materials preparation Complete plastomes from 79 Iris species were used, including 62 plastomes downloaded from NCBI database and 17 newly sequenced plastomes (Figure.1 and Table S1 ). The 17 newly collected species in this study are neither endangered nor national protected plants. All methods were carried out in accordance with relevant guidelines and regulations. The species identification process followed the opinions from three different experts(Xianfeng Jiang, Yanping Xie and Tianmeng Liu), and the related voucher specimens are stored in the publicly available herbarium(Biological Sciences Museum of Dali University). The full name of the species, the deposition number, the NCBI accession number and the collecting site were listed in the table S1 . Among all the species in this study, 49 species are naturally distributed or cultivated in China. Crocus cartwrightianus and Crocus sativus were used as outgroups. For the newly sequenced taxa, fresh and healthy leaves were collected, and the leaf sample were rapidly frozen in liquid nitrogen after carefully cleaned. The frozen samples were placed in pre-cooled polyethylene (PE) tubes and stored in -20 degrees for subsequently DNA extraction and sequencing. 1.2 Plastome sequencing, assembly, and annotation The genomic DNA was extracted from the frozen leaf samples using HiPure SF Plant DNA Mini Kit (Magen) [ 21 ], and its quality was evaluated using 1% agarose gel electrophoresis. Approximately 300 ng of high-quality DNA was used for library construction following the procedures of DNA fragmentation, end repair, 3' A-tailing, and purification. The resulting libraries were then sequenced on Illumina NovaSeq 6000 platform after quality-checked. To ensure the integrity of downstream analyses, the quality of raw sequencing reads was assessed using FastQC [ 22 ]. Raw sequencing reads were filtered using Cutadapt v1.16 [ 23 ] to remove adapters and low-quality sequences, resulting in high-quality clean reads for downstream analyses. Plastome assemblies were performed using multiple tools, including NOVOPlasty v4.2 [ 24 ] ( https://github.com/ndierckx/novoplasty ), Fast-Plast v1.2.8 [ 25 ] ( https://github.com/mrmckain/Fast-Plast ), and GetOrganelle v1.7.0 [ 26 ] ( https://github.com/Kinggerm/GetOrganelle ). The best assemblies were selected based on assembly quality metrics. Plastome annotation was conducted using PGA [ 27 ] ( https://github.com/quxiaojian/PGA ) and GeSeq [ 28 ] ( https://chlorobox.mpimp-golm.mpg.de/geseq.html ), followed by manual curation for all samples. Circular genome maps were generated using OGDRAW online tool [ 29 ] ( https://chlorobox.mpimp-golm.mpg.de/OGDraw.html ). 1.3 Comparative genome analysis 1.3.1 IR boundary analysis Comparative analysis of the IR/LSC and IR/SSC boundaries allowed us to assess the patterns of IR region expansion and contraction among different Iris plastomes. To investigate the expansion and contraction of inverted repeat (IR) regions, we compared the boundaries among the four major regions of Iris plastomes—namely, the large single copy (LSC), small single copy (SSC), and two inverted repeat regions (IRa and IRb). Specifically, the junctions of LSC/IRb (JLB), SSC/IRb (JSB), SSC/IRa (JSA), and LSC/IRa (JLA) were visualized using the online tool CPJSdraw [ 30 ] ( https://doi.org/10.7717/peerj.15326 ). 1.3.2 Codon usage analysis Codon usage bias is an important feature of biological evolution, reflecting both mutational pressure and natural selection that shape the efficiency and accuracy of gene expression. Multiple codon usage parameters were calculated using CodonW v1.4.4 [ 31 ] and the EMBOSS software suite [ 32 ]. These parameters included RSCU, total codon number (Codon No.), coding sequence length (CDS length), GC content at the second and third codon positions (GC2 and GC3), codon bias index (CBI), and effective number of codons (ENC). To further investigate codon usage patterns, 28 representative Iris species (spanning from subgen. Iris , subgen. Crossiris , subgen. Nepalensis , subgen. Limniris , subgen. Pardanthopsis , subgen. Xyridion , and others) were selected for RSCU-based heatmap visualization. Codons with the highest and lowest RSCU values across species were identified. Interpretation of RSCU values followed standard conventions: an RSCU value of 1 indicates no bias; RSCU > 1 suggests that a codon is used more frequently than expected, while RSCU < 1 indicates less frequent usage [ 31 , 33 , 34 ]. 1.3.3 Repeat sequence analysis Repeat sequences in plastid genomes are known to affect genome structural stability, recombination frequency, and evolutionary dynamics. In addition to the well-characterized inverted repeat (IR) boundary regions, other types of repetitive elements, such as short dispersed repeats (SDRs) and simple sequence repeats (SSRs), are considered important structural features that may play significant roles in plastome evolution. We performed a comprehensive analysis of SDRs and SSRs across the complete plastomes of 79 Iris species. Four types of SDRs—Forward, Reverse, Palindromic, and Complementary repeats—were identified using the online tool REPuter [ 35 ] ( https://bibiserv.cebitec.uni-bielefeld.de/reputer ). The parameters were set as follows: Hamming distance = 3, maximum number of repeats = 500, and minimum repeat length = 30 bp. Additionally, SSRs were detected using the MISA tool [ 36 ] (MISA-web, IPK Gatersleben). Six categories of microsatellites were searched, i.e., Mono-nucleotide, Di-nucleotide, Tri-nucleotide, Tetra-nucleotide, Penta-nucleotide, and Hexa-nucleotide repeats. The minimum repeat thresholds were set at 10, 5, 4, 3, 3, and 3, respectively [ 37 ]. 1.4 Selective pressure analysis The selective pressure on protein-coding genes (PCGs) of Iris plastomes was assessed based on the variation in nucleotide substitution rates [ 38 ]. Coding sequences (CDSs) were extracted, and the ratios of nonsynonymous (dN) to synonymous (dS) substitution rates (dN/dS) were estimated using the CODEML module implemented in PAML v4.9 [ 39 ]. A constraint tree generated by a genetic algorithm based on the CDS dataset was used as the input phylogeny. The parameters in the CODEML control file were set as follows: (1) F3 × 4 model for codon frequencies; (2) “model = 0” for allowing a single dN/dS value to vary among branches; (3) “cleandata = 1” to remove gaps; and (4) default settings for other parameters [ 40 ]. This analysis provided robust support for estimating lineage-specific selective constraints acting on plastid PCGs in Iris . 1.5 Comparative plastome alignment and sequence divergence analysis To evaluate the sequence divergence and the hypervariable regions among Iris species, we conducted global plastome alignments of 36 Iris species using the mVISTA software [ 41 ] in Shuffle-LAGAN alignment mode, with Iris germanica (GenBank accession: NC_062594) serving as the reference genome. This analysis revealed patterns of sequence variation and facilitated the identification of highly variable loci that may serve as potential molecular markers for species delimitation and phylogenetic reconstruction of closely related taxa. To more precisely locate the genomic regions with high mutation rates, we selected the 79 Iris plastomes to perform sliding window analysis using the Genepioneer platform ( http://cloud.genepioneer.com ) and DnaSP v6.12.03 [ 42 ]. Prior to the analysis, multiple sequence alignment of plastid genomes was carried out using MAFFT v7.505 [ 43 ] with default parameters. The sliding window analysis was conducted with a window length of 400 bp and a step size of 200 bp. Nucleotide diversity (Pi) values were calculated for each window and plotted to visualize the distribution of sequence variation across the plastomes. 1.6 Phylogenetic analysis To explore the phylogenetic relationships among Iris species, we analyzed both complete plastome and coding sequence (CDS) datasets of 79 Iris species, using C. cartwrightianus and C. sativus as outgroups. Sequence alignment was performed using MAFFT v7.505 [ 43 ], followed by manual inspection and adjustment. Non-conserved regions were trimmed using trimAl v1.2 [ 44 ]. Phylogenetic trees were reconstructed using three methods: maximum likelihood (ML) [ 45 ] in IQ-TREE v2.4.0 [ 46 ], maximum parsimony (MP) [ 47 ] in MEGA11 [ 48 ], and Bayesian inference (BI) in MrBayes v3.2.6 [ 49 ]. For ML analyses, the best-fit model (TVM + F + I + R4) was selected by ModelFinder [ 50 ] for both datasets. For BI, the best-fit model (GTR + I + G) was identified using Modeltest v3.7 [ 51 ]. Markov chain Monte Carlo (MCMC) analysis was run for 50 million generations with four chains, sampling every 1,000 generations and discarding the 25% as burn-in. Support values for both the complete plastome and CDS datasets were assessed using 1,000 ultrafast bootstrap replicates [ 52 ]. The generated phylogenetic trees were visualized using FigTree v1.4.4 [ 53 ] and iTOL [ 54 ] ( https://itol.embl.de/ ) and further beautified in Adobe Illustrator 2024 [ 55 ]. 1.7 Molecular clock dating The estimates of divergence times were based on the molecular clock theory, which assumes that codon substitutions in gene sequences occur at a nearly constant rate [ 56 ], and genetic differences between species are positively correlated with divergence times [ 57 ]. We used the plastomes of 79 Iris species for divergence time estimation, with a species tree constructed from complete plastome sequences as input. Sequences were aligned using MAFFT v. 7.526 [ 43 ], and the alignments were manually checked and adjusted to ensure accuracy. Non-conserved regions were trimmed using trimAl v. 1.2 [ 44 ]. Phylogenetic trees were inferred using IQ-TREE v. 2.4.0 [ 46 ], with the CDS and complete plastome datasets modeled under TVM + F + I + R4 as selected by ModelFinder [ 50 ]. Node support was evaluated by 1,000 rapid bootstrap replicates [ 52 ]. Divergence time calibration among species in the phylogenetic tree was based on fossil and molecular data integrated from the TimeTree database [ 58 ] ( http://timetree.org/ ). The estimated divergence time range was set within ± 0.04 Ma of the fossil- and molecular-based estimates. Fossil calibration points included the most recent common ancestor of Crocus and Iris , as well as root nodes within Iris species pairs such as Iris domestica and Iris dichotoma , Iris sanguinea and Iris pseudacorus , Iris odaesanensis and Iris minutoaurea , and Iris decora and Iris. japonica , resulting in a fossil-calibrated rooted phylogeny. Using this phylogeny, divergence times were estimated with the MCMCTree [ 52 ] method implemented in PAML 4.9 [ 39 , 59 ]. Markov chain Monte Carlo (MCMC) analyses were run for 100 million generations with four independent chains, sampling every 1,000 generations and discarding the first 25% as burn-in. Convergence of the MCMC runs was assessed using Tracer v.1.7.2 [ 60 ]. The resulting phylogeny was visualized with Figtree v.1.4.4 [ 53 ] and the online tool TVBOT [ 61 ] ( https://chiplot.online/tvbot.htm ). Result 2.1 Plastome features We analyzed the structure of 79 complete plastomes of Iris species, including 17 newly sequenced plastomes (Table 1 , Table S1 ). The plastome lengths ranged from 150,169 bp to 155,878 bp. All plastomes exhibited a typical quadripartite structure, consisting of a large single-copy (LSC) region, a small single-copy (SSC) region, and two inverted repeats (IRa and IRb) (Fig. 2 ). The overall GC content was highly conserved among all species, ranging from 37.32% to 38.23% (Table 1 ). Each plastome was annotated and manually checked, yielding 123 to 132 genes per plastome, including 84 to 88 protein-coding genes, 34 to 41 transfer RNA (tRNA) genes, and 0 to 8 ribosomal RNA (rRNA) genes (Table S2 ). Taking Iris decora as an example, its plastome contains 132 genes, of which 76 are involved in self-replication, 45 are associated with photosynthesis, and 11 are related to other functions. Among these, 20 genes are present in two copies (located in the IR regions), while the remaining 112 genes are present in a single copy. Further analysis revealed that 13 genes contain a single intron, 3 genes contain two introns, and the remaining 116 genes lack introns (Table S2 ). Table 1 Basic characteristics of the plastomes generated in this study. Species Size (bp) GC length (%) LSC length(bp) SSC length(bp) IR length(bp) Gene number Protein-coding gene number rRNA gene number tRNA gene number Total Coding LSC SSC IR Iris barbatula 153600 37.84 38 35.99 31.23 43.1 82791 18467 26171 127 86 0 41 Iris bulleyana 152198 38.08 38.16 36.26 31.86 43.07 82085 18003 26055 127 86 0 41 Iris cangshanensis 154008 37.32 37.68 35.34 30.55 42.77 83559 17937 26256 130 84 8 38 Iris chrysographes 153570 38.02 38.15 36.28 31.89 42.5 81688 16991 27445 128 87 0 41 Iris collettii 154165 37.81 37.97 35.99 31.29 42.97 83120 18567 26239 126 86 0 41 Iris decora 153787 37.83 38.02 35.98 31.22 43.08 82973 18470 26172 132 86 8 38 Iris delavayi 152397 38.05 38.17 36.23 31.8 43.08 82319 18008 26035 127 86 0 41 Iris latistyla 155878 37.75 37.95 36.02 31.24 42.41 81323 18513 28021 129 88 0 41 Iris leptophylla 155138 37.74 37.91 35.99 31.31 42.5 81761 18455 27461 128 88 0 41 Iris milesii 153202 37.89 38.13 36.14 31.42 42.98 82778 18536 25944 131 86 8 37 Iris pumila 153546 37.84 38.03 35.97 31.41 43.04 82655 18457 26217 130 86 8 36 Iris reticulata 154636 37.82 38.13 36.05 31.56 42.18 82787 15807 28021 127 86 0 41 Iris scariosa 154814 37.95 38.15 36.25 31.81 42.11 81888 16022 28452 128 87 0 41 Iris tectorum f. alba 153199 37.89 38.31 36.16 31.42 42.96 82779 18562 25929 131 88 8 35 Iris tenuifolia 150487 38.23 38.23 36.52 31.85 43.14 81136 18035 25658 130 86 8 36 Iris ventricosa 150169 38.15 38.21 36.45 32.16 42.87 84511 17302 25663 123 86 8 34 Iris wattii 153867 37.90 38.04 36.12 31.34 42.93 82298 18515 26527 132 86 8 38 2.2 IR boundary comparative analysis We analyzed four junctions of 79 Iris plastomes (Figure S1 ), i.e., JLB (junction between LSC and IRb), JSB (between SSC and IRb), JSA (between SSC and IRa), and JLA (between LSC and IRa). At the JLB junction, most species exhibited boundaries between rpl22–trnH and rpl22–rps19 . Iris leptophylla and Iris latistyla located the JLB junction in rpl16 . Iris chrysographes and Iris scarios lied the JLB boundary between rps3 and rpl22 . Iris confusa , Iris luojiensis [ 9 ], and I. japonica fall their JLB boundaries in rps19 . At the JSB junction, most boundaries were located within ycf1 or ndhF , while Iris mariae lied its JSB boundary between rps7 and trnV . Ycf1 was contracted into the IRb region in Iris dichotoma and Iris japonica . NdhF did not extend into the IRb region in Iris cangshanensis [ 8 ]. At the JSA junction, the boundary was commonly located within ycf1 . However, JSA junction lied between trnV–rps7 in Iris mariae , and lied within ndhF in Iris ventricosa and Iris missouriensis . Moreover, ycf1 in Iris missouriensis was contracted into the IRa region. At the JLA junction, the junction typically lied between trnH–psbA or rps19–psbA . In Iris wattii , the junction was located within psbA . Gene content analysis revealed that 45 species contained two copies of ycf1 , 33 species only had a single copy of ycf1 , and one species lacked ycf1 entirely. All species except Iris mariae contained the ndhF (Fig. 3 ). 2.3 Codon usage preference Codon counts of 79 Iris plastomes ranged from 27,149 in Iris goniocarpa to 29,405 in Iris scariosa (Table S3 ). Codon usage frequencies were largely conserved across species (Table S4 ). Among the 20 amino acids encoded by 64 codons, only methionine (Met) and tryptophan (Trp) were represented by a single codon (Fig. 4 ). The relative synonymous codon usage (RSCU) values across all species showed low overall variation (Table S4 ). The three codons with the highest RSCU values were CGA (Arg), CUG (Leu), and GUU (Val), and the lowest values were UUG (Leu), UUA (Leu), and AGG (Arg) (Table S4 , Figure S2 ). A histogram of CBI values across Iris species revealed a narrow distribution, with the majority of genes exhibiting values tightly clustered between − 0.112 and − 0.109. This indicates a consistent pattern of mild codon usage bias across genes. No genes showed positive CBI values, suggesting an overall absence of strong preference for optimal codons in the examined dataset (Figure S3 ). Further analysis revealed that codons ending in T or A generally had RSCU values > 1, while those ending in C or G typically had RSCU values < 1 (Table S4 ). These findings suggest a bias toward the use of T and A at the third codon position [ 62 ], a pattern commonly observed in angiosperm plastid genomes [ 63 ]. Moreover, GC content analysis showed that GC2 values were significantly higher than GC3 values across all Iris species, indicating a stronger preference for G/C bases at the second codon position (Table S4 ). 2.4 Repeat analysis The number of SDRs varied markedly among species, ranging from 28 in Iris speculatrix to 200 in Iris cangshanensis (Fig. 5 A; Table S5 ). Classification of repeat types revealed that palindromic repeats (~ 48.0%) and forward repeats (~ 42.4%) were the most, while reverse (~ 7.5%) and complement repeats (~ 2.1%) were relatively rare (Fig. 5 A; Table S5 ). For the SSRs, the number detected per plastome ranged from 31 in Iris halophila to 74 in Iris germanica (Fig. 5 B; Table S6 ). Among the identified SSR types, Mono-nucleotide repeats were the most abundant (~ 60.2%), followed by Di-nucleotide repeats (~ 21.2%). Hexa-nucleotide repeats were the least frequent, accounting for only ~ 0.5% of the total SSRs (Fig. 5 B; Table S6 ). 2.5 Selective pressure analysis The dN/dS ratio is a widely used indicator of selection: dN/dS = 1 suggest neutral evolution, dN/dS 1 imply diversifying (positive) selection. Across the genes analyzed, dN values ranged from 0 to 0.0430, and dS values from 0.0062 to 0.1239, with corresponding dN/dS ratios spanning 0 to 0.9215 (Fig. 6 ; Table S7 ). cemA exhibited the highest nonsynonymous substitution rate (dN = 0.0430), while petD had the highest synonymous rate (dS = 0.1239). Notably, ycf2 showed the highest dN/dS ratio (0.9215) (Fig. 6 ; Table S7 ). In contrast, psbT (dN/dS = 0), psbD (dN/dS = 0.0033), and atpH (dN/dS = 0.0033) exhibited the lowest dN/dS values (Fig. 6 ; Table S7 ). 2.6 Comparative plastome alignment and detection of hypervariable regions The global alignment of 36 representative taxa revealed pronounced sequence divergence in both coding and non-coding regions. Several protein-coding genes— ycf1 , rps16 , and ndhF —exhibited substantial variation. Additionally, intergenic regions such as trnS-trnG , trnD-trnY , petA-psbJ , ndhF-rpl32 , rps7-trnV , rpl22-rps19 , and rbcL-accD showed high sequence divergence (Figure S4 A, B). Pi values across the plastomes of 79 Iris species ranged from 0.00043 to 0.06705, with an average nucleotide diversity of 0.019. Comparative analysis of genome structure revealed that the SSC region exhibited significantly higher genetic variation than LSC and IR regions (Fig. 7 ). Ycf1 (Pi = 0.06705, 0.06510), ndhF (Pi = 0.06018), and intergenic spacer ccsA-psaC (Pi = 0.04704) harbored the highest nucleotide variation (Fig. 7 ). These results underscored the SSC as the most variable plastome region across Iris species. In addition to these hypervariable loci in the SSC, several regions within the LSC also exhibited elevated levels of genetic diversity, including the intergenic region trnS-trnG (Pi = 0.04420), the coding gene rpl16 (Pi = 0.03921), and the spacer petA-psbJ (Pi = 0.03572) (Fig. 7 ; Table 2). Table 2 Seven hypervariable region genes Genes Pi (Nucleotide diversity) S (Number of polymorphic sites) Position ycf1 0.06705 264 SSC ycf1 0.06510 277 SSC ndhF 0.06018 244 SSC ccsA-ndhD 0.04704 264 SSC trnS-trnG 0.04420 190 LSC rpl16 0.03921 125 LSC petA-psbJ 0.03572 139 LSC 2.7 Phylogenetic relationships Both Maximum Likelihood (ML), Maximum Parsimony (MP), and Bayesian Inference (BI) analyses produced highly congruent phylogenetic trees, supporting an almost identical overall topology (Fig. 8 ; Figure S5 ). Both CDS and complete plastome datasets yielded largely concordant topologies, with only two inconsistencies observed (Figure S5 ). Overall, the complete plastome dataset provided better phylogenetic resolution than the CDS dataset, as indicated by higher bootstrap support values (Fig. 8 ; Figure S5 ). Of particular note, Iris luojiensis [ 9 ], a species recently described in June 2024, was clustered with subgen. Crossiris across all phylogenetic inference methods. It received strong nodal support (ML = 99, MP = 98, BI = 1.00), highlighting the robustness and taxonomic stability of its phylogenetic placement (Fig. 8 ; Figure S5 ). In contrast, Iris cangshanensis [ 64 ] was resolved as a strongly supported monophyletic lineage, receiving full support from all three analytical frameworks (ML = 100, MP = 100, BI = 1.00). This finding highlights its substantial phylogenetic divergence from other Iris species and supports its recognition as a distinct evolutionary lineage (Fig. 8 ; Figure S5 ). The phylogenetic positions of species in other lineages remained stable, and the tree constructed using the complete plastome dataset exhibited significantly stronger support than that based on CDSs (Fig. 8 ; Figure S5 ). A comparative analysis was conducted on 79 Iris species focusing on two morphological traits: the type of appendages on the falls and the form of underground storage organs, indicating the original traits of Iris being bearded and rhizomelic (Fig. 8 ). 2.8 Divergence time estimation The initial divergence between Iris and Crocus was estimated at approximately 49.00 million years ago (Ma) during the Eocene epoch, with a 95% highest posterior density (HPD) interval of 44.55–52.41 Ma (Fig. 9 ). Within subgen. Crossiris , multiple lineage divergences occurred during the Miocene to Pliocene epochs, with estimated dates at 21.43 Ma (95% HPD: 16.02–27.54 Ma), 4.31 Ma (1.77–7.16 Ma), 4.21 Ma (1.63–7.19 Ma), and 2.82 Ma (0.63–5.63 Ma). Divergence within subgen. Iris was estimated at ~ 10.37 Ma (95% HPD: 5.47–20.00 Ma, Miocene), with a much more recent event at 0.02 Ma (95% HPD: 0–0.08 Ma, Quaternary), indicating the coexistence of both ancient and recently evolved lineages. Major divergence events in subgen. Nepalensis were dated to 13.56 Ma (95% HPD: 8.43–18.93 Ma, Miocene) and 2.41 Ma (95% HPD: 0.36–4.68 Ma, Quaternary), suggesting substantial lineage diversification across geological timescales. Additionally, Subgen. Pardanthopsis and Subgen. Hermodactyloides exhibited more recent divergence times of 0.40 Ma (95% HPD: 0.21–0.62 Ma) and 0.24 Ma (95% HPD: 0.05–0.45 Ma), respectively. The primary divergence event of Subgen. Scorpiris could date back to 6.66 Ma (95% HPD: 0.37–9.77 Ma) within Miocene epoch (Fig. 9 ). For recently described species, Iris cangshanensis [ 64 ] was estimated to have originated approximately 44.40 Ma (95% HPD: 37.03–51.25 Ma, Eocene), while Iris luojiensis [ 9 ] diverged at around 3.65 Ma (95% HPD: 1.24–6.33 Ma, Pliocene). Overall, divergence times among major Iris species ranged from 0.02 to 25.00 Ma, primarily within the Miocene to Quaternary periods, indicating multiple episodes of lineage radiation and ecological adaptation throughout the Cenozoic. Discussion Plastome characteristics of Iris The plastome structure across 79 Iris spcecies is largely conserved. All plastomes exhibit typical quadripartite structure, consisting of a large single-copy (LSC) region, a small single-copy (SSC) region, and two inverted repeats (IRa and IRb) (Fig. 2 ). Such structure is similar to that reported among other studies on Iris [ 65 , 66 ]. Similar to most angiosperm plastomes, the conservation of plastome structure is likely consistent to the requirement of maintaining the stability of plastome functionality [ 67 ]. 123 to 132 genes are detected per plastome of Iris , including 84 to 88 protein-coding genes. 76 among these genes are involved in self-replication, 45 are associated with photosynthesis, and 11 are related to other functions. Expansions and contractions are observed in the IR regions of the 79 Iris plastomes. IR regions of angiosperm plastomes generally begin near rps19 gene and end consistently downstream at trnN-GUU or ycf1 gene [ 68 ]. While IR expansion has been documented in specific lineages, usually within the LSC region [ 68 ]. We discover four junctions: JLB (junction between LSC and IRb), JSB (between SSC and IRb), JSA (between SSC and IRa), and JLA (between LSC and IRa) in this study. These findings suggest that IR expansions are independent events in the genus Iris . An analysis of different SSR repeat types reveals that mononucleotide repeats are the most prevalent (~ 60.2%), followed by Di-nucleotide repeats (~ 21.2%). SSR numbers varied slightly across the 79 Iris taxa, ranging from 31 to 74. The predominance of palindromic and forward repeats suggests their potential roles in maintaining plastome structural stability and facilitating intragenomic recombination. This study aligns with other plastome studies of Iris [ 65 , 66 ]. Together, these results characterize the distribution patterns of repetitive elements in Iris plastid genomes and provide a valuable basis for future evolutionary studies and the development of plastid-based molecular markers. Factors influencing codon usage include genome size, base mutation, genetic drift, natural selection, gene expression level, and protein structure [ 69 ]. Synonymous codons arise from mutations and may differ in their usage frequency, which can be quantified by relative synonymous codon usage (RSCU), a measure that reveals codon usage preferences among genes [ 70 ]. Codon counts of 79 Iris plastomes range from 27,149 in Iris goniocarpa to 29,405 in Iris scariosa (Table S3 ). Codon usage frequencies are largely conserved across species (Table S4 ). Among the 20 amino acids encoded by 64 codons, only methionine (Met) and tryptophan (Trp) are represented by a single codon (Fig. 4 ). 65 genes are identified to undergo negative selection with the corresponding dN/dS ratios ranging from 0 to 0.9215. Most of the negative selective genes are involved in self-replication, photosynthesis, and protein synthesis. Among them, ycf2 shows the highest dN/dS ratio (0.9215), suggesting it undergoes relaxed purifying selection pressure with elevated evolutionary rate, potentially due to relaxed functional constraints or lineage-specific adaptive evolution (Fig. 6 ; Table S7 ). In contrast, psbT (dN/dS = 0), psbD (dN/dS = 0.0033), and atpH (dN/dS = 0.0033) exhibited the lowest dN/dS values, indicating they undergo strong purifying selection with high evolutionary conservation. These negative selected genes are known to play critical roles in photosynthetic complexes, which may account for their functional constraint (Fig. 6 ; Table S7 ). Collectively, these findings highlight the heterogeneity of selective pressures across Iris plastid genes and provide molecular insights into the evolutionary history and adaptive divergence of genus Iris . Phylogenetic analysis and adaptive evolution Compared to previous phylogenetic studies on Iris [ 14 , 16 , 65 ], this study is the most comprehensive one to analyze Iris phylogenetic relationships with density sampling using plastome-scale sequences. Our plastome tree provides a more robust phylogenetic framework for the genus Iris compared to the studies based on single or multiple locus DNA sequences, with major nodes showing strong support (i.e., PP = 1.00 and BS ≥ 80). The results further confirmed that whole plastome sequencing can enhance the phylogenetic resolution within a given lineage. Most Iris species were resolved as clustered into a monophyletic based on the plastome sequence data, except for Iris cangshanensis , which is inferred to be a monophyletic lineage that locates at the basal of the Iris clade. It implies that Iris cangshanensis may have a more complex taxonomic relationship and evolutionary history than we previously thought [ 8 , 64 ]. This study is the first to analyze the maternal evolutionary history of the genus Iirs based on a large dataset of plastomes. Notably, our plastome-based phylogenomic tree indicates three major clades in Iris with high credibility support (Fig. 8 ). The clade A composes of subgenus Iris and subgenus Pardanthopsis . The clade B includes subgenus Scorpiris , subgenus Crossiris and subgenus Nepalensis . The clade C composes of subgenus Limmirs , Iris scariosa , Iris reticulate , Iris halophila and Iris speculatrix . This result is partially consistent with the previous phylogeny tree based on 19 Iris plastomes [ 65 ]. As for the previous phylogeny studies using cpDNA sequences on Iris by Wilson [ 18 , 26 ] and Guo et al. [ 16 ], some of their results are also consistent with this study. For examples, Wilson's findings indicate that the subgenus Limmirs is an independent branch, while Guo's results suggest that subgenus Scorpiris , subgenus Crossiris and subgenus Nepalensis form a clade. Hybridization and introgression commonly occur among Iris with sympatric distributions [ 71 , 72 ]. When interspecific gene flow is asymmetric, one parental species may experience assimilation of its nuclear genome, while its maternal plastome is retained in the populations. This phenomenon is commonly referred to as the introgression-induced chloroplast capture [ 73 ]. Introgression-induced chloroplast capture has been identified as a mechanism that can distort phylogenetic relationships, often resulting in geographic clustering of introgressed taxa. Natural hybridization and introgression are commonly observed among the species within Iris [ 71 , 72 , 74 ]. The present phylogenetic work on Iris revealed a secondary increase in the speciation rate of Iris during Oligocene and Miocene, suggesting that interspecific hybridization may have occurred during the early stages of its diversification (Fig. 9 ). Incomplete lineage sorting among taxa is often associated with radiations [ 75 ]. Accordingly, the possibility of incomplete lineage sorting causing cytonuclear discordance cannot be totally discounted. Furthermore, pollen and seed dispersal are critical determinants of gene flow [ 76 ]. Gene flow via pollen is significantly greater than that occurring via seeds, leading to broader genetic exchange for the nuclear genome compared to the plastome. Differences between seed- and pollen-mediated gene flow can result in cytonuclear discordance in phylogenetic studies [ 77 ]. Iris species are primarily pollinated by insects in natural habitats [ 74 ], thus achieve the long-distance transmission possibility of pollen enhances gene flow among populations. In contrast, the seed dispersal of Iris is more limited [ 78 , 79 ]. The contrasting patterns of pollen- and seed-mediated gene flow among the ancestral populations could contribute to the cyto-nuclear discordance observed in Iris [ 71 , 80 ]. We found significantly nuclear-cytoplasmic conflict of genus Iris (unpublished data), but all the hypotheses above should be tested concisely in future studies. Correct phylogeny and divergence-time estimation are essential for evolutionary history study. This study is the first to conduct divergence-time estimation on Iris . An appropriate molecular markers selected is of great concern when inferring a phylogeny of targeted taxa, as the selected markers can strongly affect overall topology and divergence time estimates. With a complete chloroplast gene set, we can choose suitable genes to facilitate and optimize divergence-time estimation. The present divergence-time estimation indicates that the crown node age of Iirs is estimated at 49.00 million years ago (Ma). Iris cangshanensis diversifed from major Iris clade at 44.40 Ma. The differentiation of subgenus Limniris clade between other groups of the Iris occurred at Oligocene (29.82 Ma), and the clade B radiated at Miocene (20.19 Ma) (Fig. 9 ). This study conducts certain investigations into the morphological evolution of Iris genus. We mainly focus on two key traits of Iris , i.e., the morphology of the underground organs and the form of the nectary guide. We classify the nectar guide on the falls into three types, i.e., beards, crests, and those with spots, and divide the root morphology into tuberous, bulbous and rhizomelic (Fig. 8 ). The present results indicate that the beard or crest on falls occurred independently multiple times in subgen. Iris , subgen. Nepalensis , and subgen. Crossiris , respectively. As for the root morphology evolution in Iris , the present results indicate that rhizome might be the original trait of Iris and independently evolved into bulbous (Subgenus Scorpiris ) or tuberous (Subgenus Nepalensis ). The plastome-based phylogenetic framework does not align with key taxonomical groupings based on the underground organ or the appendage on fall (Fig. 8 ), indicating that these morphological traits are limited phylogenetically informative at the subgenera ranks in some subgenera classification of the genus Iris . The paraphyletic pattern of beard species on the plastome trees may indicate a consequence of convergent adaptation to cope with animal predation [ 81 , 82 ], and the independently origin of the bulbous or tuberous root may indicate the adaptation for the special external inorganic environment [ 83 ]. Conclusions In this study, we sequence and assemble the complete chloroplast genomes of 17 Iris specie, and compare the structure of 79 Iris plastomes by adding 62 published samples. Phylogenetic analysis based on the chloroplast genome supported part of the previous subgenera taxonomic treatment study using morphological characteristics and fragments sequences. Divergence time analysis revealed that Iris originated at early Eocene and diversified at early Oligoceae. Beard or crest species, in combination with the rhizomelic roots, might be the original traits of genus Iris , and independently evolve into beardless, tuberous and bulbous. Overall, this study demonstrates that the whole chloroplast genome sequences display variable information to resolve phylogenetic relationships in this genus. Declarations Ethics approval and consent to participate The collecting of all samples in this study followed the Regulations on the Protection of Wild Plants of China, 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. All methods were carried out in accordance with relevant guidelines and regulations. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding The present research was funded by the National Natural Science Foundation of China (Grant No. 32460335), the Foundation of Yunnan Province Science and Technology Department (Grant No. 202305AM070003), and the National Natural Science Foundation of China (32100169). Author Contribution Jinfeng Liu, Xin Jin and Xianfeng Jiang collected samples, Jinfeng Liu and Xingtang Du were responsible for the overall analysis of the data of the article, Yanping Xie provided comprehensive guidance for the overall analysis, and Xianfeng Jiang was responsible for the writing and revision of the article. Acknowledgements Not applicable. 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14:14:27","extension":"xml","order_by":61,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":203467,"visible":true,"origin":"","legend":"","description":"","filename":"c9c887759f4744e58922371f5e0e3aa21structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/3fdce98e1792b792a32cc3d8.xml"},{"id":97668208,"identity":"09130517-9fdc-4af3-baaa-22b5ec6353c3","added_by":"auto","created_at":"2025-12-08 09:25:02","extension":"html","order_by":62,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":226471,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/51b0f841f6f700a7bbb98878.html"},{"id":97668703,"identity":"74e307f8-6e25-47ea-a771-c2530804f7f6","added_by":"auto","created_at":"2025-12-08 09:26:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7124344,"visible":true,"origin":"","legend":"\u003cp\u003eSome of the \u003cem\u003eIris\u003c/em\u003especies collected and analyzed in this study. (A) \u003cem\u003eIris tectorum\u003c/em\u003e var. \u003cem\u003ealba\u003c/em\u003e(B) \u003cem\u003eIris collettii\u003c/em\u003e (C)\u003cem\u003e Iris latistyla \u003c/em\u003e(D) \u003cem\u003eIris bulleyana\u003c/em\u003e (E) \u003cem\u003eIris cangshanensis \u003c/em\u003e(F) \u003cem\u003eIris delavayi\u003c/em\u003e(G) \u003cem\u003eIris ventricosa\u003c/em\u003e (H) \u003cem\u003eIris leptophylla\u003c/em\u003e (I) \u003cem\u003eIris tenuifolia\u003c/em\u003e (J) \u003cem\u003eIris wattii \u003c/em\u003e(K)\u003cem\u003e Iris barbatula\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/f1f45c3e72a6b758e1274100.jpg"},{"id":97454676,"identity":"54b44037-c635-435a-adf1-3c7030b1ca78","added_by":"auto","created_at":"2025-12-04 14:14:25","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2052162,"visible":true,"origin":"","legend":"\u003cp\u003eCircular gene map of the \u003cem\u003eIris\u003c/em\u003e plastome. From the center outward, the central part shows the range of plastome length. The first track indicates the GC content of the genome. The dark gray in the first track represents the GC content, and the light gray indicates the AT content. The legends in different colors at the bottom left represent genes with different functions\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/8a2ae6010e000d7f28be82a1.jpg"},{"id":97668907,"identity":"c2518672-cf17-446c-863c-57cf14cdfb07","added_by":"auto","created_at":"2025-12-08 09:26:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3551370,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of four IR borders among \u003cem\u003eIris\u003c/em\u003e. The adjacent border genes are indicated by boxes with gene names inside them and the same color indicates the same gene. The phylogenetic tree was inferred from comlete plastome dataset\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/deaedce84658b0968f155a45.jpg"},{"id":97454682,"identity":"33de5345-35c7-4329-8ece-6b79f711f3ac","added_by":"auto","created_at":"2025-12-04 14:14:25","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":735402,"visible":true,"origin":"","legend":"\u003cp\u003eCodon content of 20 amino acids and stop codon in \u003cem\u003eIris\u003c/em\u003eplastomes. The relative synonymous codon usage (RSCU) values are shown on the y-axis\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/f756e7ae0b1f94de298ca879.jpg"},{"id":97454688,"identity":"06e77c8c-4faa-493a-b3d7-4ccbcde49029","added_by":"auto","created_at":"2025-12-04 14:14:25","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6261697,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of repeat elements in \u003cem\u003eIris\u003c/em\u003e plastomes. \u003cstrong\u003eA\u003c/strong\u003e Frequency and average proportion of four types of short dispersed repeats (SDRs). \u003cstrong\u003eB\u003c/strong\u003e Frequency and average proportion of six simple sequence repeats (SSRs) types.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/668cc6ec8b08628bd13fd3bc.jpg"},{"id":97668632,"identity":"3e839216-0b9a-4d32-87f1-86a2186d2c73","added_by":"auto","created_at":"2025-12-08 09:25:52","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":804386,"visible":true,"origin":"","legend":"\u003cp\u003eThe synonymous (dS), nonsynonymous (dN) substitution rates and dN/dS of plastid protein-coding genes (PCG) in \u003cem\u003eIris\u003c/em\u003e plastomes\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/3620c5e4c27f53c31626a0d7.jpg"},{"id":97669005,"identity":"b85070b1-2592-41ab-8755-9a959aa6fee9","added_by":"auto","created_at":"2025-12-08 09:26:57","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1835317,"visible":true,"origin":"","legend":"\u003cp\u003eWindow analysis of \u003cem\u003eIris\u003c/em\u003e plastomes (window length: 400bp; step size: 200bp)\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/caac2a793abe35596e121dee.jpg"},{"id":97669185,"identity":"c3e9c59c-f3bd-4331-9420-aaf2e081d07f","added_by":"auto","created_at":"2025-12-08 09:27:32","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":6906217,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum Likelihood and Maximum Parsimony (MP) cladogram (A) and phylogram (B) of \u003cem\u003eIris\u003c/em\u003einferred from complete plastomes using IQTREE v2.4.0 and NEGA11. Node supports are indicated by ML/MP bootstrap values (BS) and posterior probabilities (PP) calculated froxm MrBayes, with \"-\" denoting BS values below 50% or absent PP values, The classification according to Mathew (1989) and Zhao Yutang (1985)\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/764845cbc5d1a67b6d0b6007.jpg"},{"id":97668737,"identity":"f909c189-9460-4010-8e77-50fb8b4ea6a4","added_by":"auto","created_at":"2025-12-08 09:26:14","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2669235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDivergence times for Iris from MCMCTree based on the \u003c/em\u003ecomplete plastome\u003cem\u003e. Mean divergence times of nodes are shown at the nodes, and green bars correspond to the 95% highest posterior density (HPD). Outgroup taxa are marked with dark gray bars\u003c/em\u003e\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/f7b43623b9e5706f56e209ce.jpg"},{"id":97893182,"identity":"7135bd0a-9d46-4571-9310-eff2f1ca0434","added_by":"auto","created_at":"2025-12-10 15:28:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":33393400,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/7c18580a-e1e2-40cd-be10-a30c49b0b8e1.pdf"},{"id":97667371,"identity":"8125d4bb-bcc1-4861-b00b-cb79aca21860","added_by":"auto","created_at":"2025-12-08 09:23:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2456027,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1S5.docx","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/9d836a2d3f7d6af95707ed89.docx"},{"id":97454684,"identity":"80dd112d-9a29-4361-b5a4-2f07c34188de","added_by":"auto","created_at":"2025-12-04 14:14:25","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":22365,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/775f39420f6d50b1e177db5e.docx"},{"id":97454678,"identity":"ee682dea-17c0-4d0c-a28c-b4d5b9bd1fc1","added_by":"auto","created_at":"2025-12-04 14:14:25","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":16241,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/d788732abba382f37c4ccc3c.docx"},{"id":97668502,"identity":"7676b426-df6e-47b2-97b4-65646e21564d","added_by":"auto","created_at":"2025-12-08 09:25:42","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":28206,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/6c2c03751e5ccca3ca6d02a4.docx"},{"id":97667988,"identity":"05413ee7-cb44-4912-840a-50be0dd2cc02","added_by":"auto","created_at":"2025-12-08 09:24:36","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16866,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/bb4fd2bd22df68e6dbc9b218.docx"},{"id":97454694,"identity":"3b7dd2c6-ab7e-46a9-9c9c-96bd53af40c4","added_by":"auto","created_at":"2025-12-04 14:14:25","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":28923,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.docx","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/e3cc9f676fdab897fc56e3d6.docx"},{"id":97454690,"identity":"fbeff6b6-dcfc-4545-9f04-1c6790203ebf","added_by":"auto","created_at":"2025-12-04 14:14:25","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":31654,"visible":true,"origin":"","legend":"","description":"","filename":"TableS6.docx","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/6e0481a0daa73e162a01f029.docx"},{"id":97454704,"identity":"84928a93-592a-4a1b-96cc-461e4a3f1e39","added_by":"auto","created_at":"2025-12-04 14:14:25","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":20755,"visible":true,"origin":"","legend":"","description":"","filename":"TableS7.docx","url":"https://assets-eu.researchsquare.com/files/rs-7909828/v1/de1d4a541f01878ca629af8e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Plastome Analysis and Phylogenetic Reconstruction of Iris Speci es in China","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIrises are a group of perennial herbaceous plants widely distributed across temperate regions of Eurasia [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. There are around 300 \u003cem\u003eIris\u003c/em\u003e species globally, with Europe to Central Asia being one of the major diversity center in the world. \u003cem\u003eIris\u003c/em\u003e is highly valued for its striking beauty and economic uses [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. As a group of well-known horticultural plants, it has a very long cultivation history across most civilization of the world [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The cultivation of Irises could date back to ancient Egypt, Asia Minor, and ancient India as early as 3000 BCE [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In ancient Greece, \u003cem\u003eIris\u003c/em\u003e is regarded as the embodiment of the divine. The name of the genus, \u003cem\u003eIris\u003c/em\u003e, comes from the Greek goddess of the rainbow, who served as a messenger between gods and mortals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. China is home to nearly 70 \u003cem\u003eIris\u003c/em\u003e species, accounting for nearly one-fifth of world's Irises species, primarily distributed across southwest, northwest, and northeast China [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Until now, a series of newly described \u003cem\u003eIris\u003c/em\u003e species continue to be found and documented in China [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In Flora of China, Irises in China were assigned into six subgenera and 7 sections based on the geographical distribution, morphological characteristics and genetic relationships [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], largely follows Rodionenko's system [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIrises produce conspicuous flowers that are characterized by colourful perianth whorls, drooping lower petals (falls), elevated upper petals (standards), nectary guides (beard, crest or stain), and petaloid style branches [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Generally speaking, the flowers of wild Irises are usually blue, yellow or red [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The basal leaves of \u003cem\u003eIris\u003c/em\u003e form a fan-shaped rosette, with a few reduced cauline leaves emerging from the reproductive stem. \u003cem\u003eIris\u003c/em\u003e species exhibit a variety of underground organs, including rhizomes, bulbs, tuberous roots, and stolons. The crest on the falls of \u003cem\u003eIris\u003c/em\u003e displays high diversity that may be bearded or beardless [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Taxonomically, \u003cem\u003eIris\u003c/em\u003e is divided into numerous subgenera primarily based on the traits of underground organs, crest on falls, and seed arils [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe crests on falls play crucial role in the taxonomic subgenera classification of \u003cem\u003eIris\u003c/em\u003e. The first comprehensive systematic study of \u003cem\u003eIris\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] categorized approximately 140 species into 12 sections, with crested species placed in three sections. Six crested species with stout rhizomes were referred to sect. \u003cem\u003eEvansia\u003c/em\u003e, and the crested species with fleshy rootstocks were assigned to sect. \u003cem\u003eNepalensis\u003c/em\u003e. Lawrence [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] downgraded sect. \u003cem\u003eEvansia\u003c/em\u003e to subsection and raised sect. \u003cem\u003eNepalensis\u003c/em\u003e to subg. \u003cem\u003eNepalensis\u003c/em\u003e. In 1987, Rodionenko [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] promoted subsect. \u003cem\u003eEvansia\u003c/em\u003e to subg. \u003cem\u003eCrossiris\u003c/em\u003e. Mathew [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] adopted Lawrence\u0026rsquo;s [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] taxonomic definitions and elevated the subsection \u003cem\u003eEvansia\u003c/em\u003e to sectional level, placing it within subg. \u003cem\u003eLimniris\u003c/em\u003e, as sect. \u003cem\u003eLophiris\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eUnderground organ is one of the other important classification criterions in \u003cem\u003eIris\u003c/em\u003e. Dykes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] proposed that rhizomes and bulbs are the original underground traits of \u003cem\u003eIris\u003c/em\u003e. These two traits firstly appeared in subgenus \u003cem\u003eLimniris\u003c/em\u003e, and then evolve to other species of \u003cem\u003eIris\u003c/em\u003e. Taking \u003cem\u003eIris grant-duffii\u003c/em\u003e as an example (a species which possesses bulbous root and rhizomes, simultaneously), he also proposed that the bulbous root was more original, and subsequently replaced by rhizomes. Lawrence [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] classified the bulbous species of \u003cem\u003eIris\u003c/em\u003e into two subgenera, the subgenus \u003cem\u003eXiphium\u003c/em\u003e has bulbous roots but lacking of fleshy roots, and subgenus \u003cem\u003eScorpiris\u003c/em\u003e has both bulbous roots and fleshy roots.\u003c/p\u003e\u003cp\u003eThe genus \u003cem\u003eIris\u003c/em\u003e has long been recognized as monophyletic group. However, various phylogeny study indicated that whether it\u0026rsquo;s a monophyly still remain controversial [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For example, Wilson [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] reconstructed the cladogram of \u003cem\u003eIris\u003c/em\u003e based on three chloroplast DNA fragments for 104 \u003cem\u003eIris\u003c/em\u003e species indicated that three monotypic genera Belamcanda, Pardanthopsis, and Hermodactylus clustered within the same clade with \u003cem\u003eIris\u003c/em\u003e. Furthermore, the inter-specific relationships within \u003cem\u003eIris\u003c/em\u003e are much more complex [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. For examples, except for the subgen. \u003cem\u003eNepalensis\u003c/em\u003e and the subgen. \u003cem\u003eXiphium\u003c/em\u003e, all the subgenera of \u003cem\u003eIris\u003c/em\u003e were identified as non-monophyletic [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. As chloroplast genome could offer significantly more abundant information compared to traditional PCR-amplified fragments [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This study will collect and sequence the chloroplast genomes of 79 Irises to investigate the genomic characteristics and evolutionary relationship within this group.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e1.1 Data and plant materials preparation\u003c/h2\u003e\u003cp\u003eComplete plastomes from 79 \u003cem\u003eIris\u003c/em\u003e species were used, including 62 plastomes downloaded from NCBI database and 17 newly sequenced plastomes (Figure.1 and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The 17 newly collected species in this study are neither endangered nor national protected plants. All methods were carried out in accordance with relevant guidelines and regulations. The species identification process followed the opinions from three different experts(Xianfeng Jiang, Yanping Xie and Tianmeng Liu), and the related voucher specimens are stored in the publicly available herbarium(Biological Sciences Museum of Dali University). The full name of the species, the deposition number, the NCBI accession number and the collecting site were listed in the table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Among all the species in this study, 49 species are naturally distributed or cultivated in China. \u003cem\u003eCrocus cartwrightianus\u003c/em\u003e and \u003cem\u003eCrocus sativus\u003c/em\u003e were used as outgroups. For the newly sequenced taxa, fresh and healthy leaves were collected, and the leaf sample were rapidly frozen in liquid nitrogen after carefully cleaned. The frozen samples were placed in pre-cooled polyethylene (PE) tubes and stored in -20 degrees for subsequently DNA extraction and sequencing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e1.2 Plastome sequencing, assembly, and annotation\u003c/h2\u003e\u003cp\u003eThe genomic DNA was extracted from the frozen leaf samples using HiPure SF Plant DNA Mini Kit (Magen) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and its quality was evaluated using 1% agarose gel electrophoresis. Approximately 300 ng of high-quality DNA was used for library construction following the procedures of DNA fragmentation, end repair, 3' A-tailing, and purification. The resulting libraries were then sequenced on Illumina NovaSeq 6000 platform after quality-checked. To ensure the integrity of downstream analyses, the quality of raw sequencing reads was assessed using FastQC [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Raw sequencing reads were filtered using Cutadapt v1.16 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] to remove adapters and low-quality sequences, resulting in high-quality clean reads for downstream analyses. Plastome assemblies were performed using multiple tools, including NOVOPlasty v4.2 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/ndierckx/novoplasty\u003c/span\u003e\u003cspan address=\"https://github.com/ndierckx/novoplasty\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Fast-Plast v1.2.8 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/mrmckain/Fast-Plast\u003c/span\u003e\u003cspan address=\"https://github.com/mrmckain/Fast-Plast\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and GetOrganelle v1.7.0 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/Kinggerm/GetOrganelle\u003c/span\u003e\u003cspan address=\"https://github.com/Kinggerm/GetOrganelle\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The best assemblies were selected based on assembly quality metrics. Plastome annotation was conducted using PGA [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/quxiaojian/PGA\u003c/span\u003e\u003cspan address=\"https://github.com/quxiaojian/PGA\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and GeSeq [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chlorobox.mpimp-golm.mpg.de/geseq.html\u003c/span\u003e\u003cspan address=\"https://chlorobox.mpimp-golm.mpg.de/geseq.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), followed by manual curation for all samples. Circular genome maps were generated using OGDRAW online tool [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chlorobox.mpimp-golm.mpg.de/OGDraw.html\u003c/span\u003e\u003cspan address=\"https://chlorobox.mpimp-golm.mpg.de/OGDraw.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e1.3 Comparative genome analysis\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e1.3.1 IR boundary analysis\u003c/h2\u003e\u003cp\u003eComparative analysis of the IR/LSC and IR/SSC boundaries allowed us to assess the patterns of IR region expansion and contraction among different \u003cem\u003eIris\u003c/em\u003e plastomes. To investigate the expansion and contraction of inverted repeat (IR) regions, we compared the boundaries among the four major regions of \u003cem\u003eIris\u003c/em\u003e plastomes\u0026mdash;namely, the large single copy (LSC), small single copy (SSC), and two inverted repeat regions (IRa and IRb). Specifically, the junctions of LSC/IRb (JLB), SSC/IRb (JSB), SSC/IRa (JSA), and LSC/IRa (JLA) were visualized using the online tool CPJSdraw [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7717/peerj.15326\u003c/span\u003e\u003cspan address=\"10.7717/peerj.15326\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e1.3.2 Codon usage analysis\u003c/h2\u003e\u003cp\u003eCodon usage bias is an important feature of biological evolution, reflecting both mutational pressure and natural selection that shape the efficiency and accuracy of gene expression. Multiple codon usage parameters were calculated using CodonW v1.4.4 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and the EMBOSS software suite [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These parameters included RSCU, total codon number (Codon No.), coding sequence length (CDS length), GC content at the second and third codon positions (GC2 and GC3), codon bias index (CBI), and effective number of codons (ENC). To further investigate codon usage patterns, 28 representative \u003cem\u003eIris\u003c/em\u003e species (spanning from subgen. \u003cem\u003eIris\u003c/em\u003e, subgen. \u003cem\u003eCrossiris\u003c/em\u003e, subgen. \u003cem\u003eNepalensis\u003c/em\u003e, subgen. \u003cem\u003eLimniris\u003c/em\u003e, subgen. \u003cem\u003ePardanthopsis\u003c/em\u003e, subgen. \u003cem\u003eXyridion\u003c/em\u003e, and others) were selected for RSCU-based heatmap visualization. Codons with the highest and lowest RSCU values across species were identified. Interpretation of RSCU values followed standard conventions: an RSCU value of 1 indicates no bias; RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1 suggests that a codon is used more frequently than expected, while RSCU\u0026thinsp;\u0026lt;\u0026thinsp;1 indicates less frequent usage [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e1.3.3 Repeat sequence analysis\u003c/h2\u003e\u003cp\u003eRepeat sequences in plastid genomes are known to affect genome structural stability, recombination frequency, and evolutionary dynamics. In addition to the well-characterized inverted repeat (IR) boundary regions, other types of repetitive elements, such as short dispersed repeats (SDRs) and simple sequence repeats (SSRs), are considered important structural features that may play significant roles in plastome evolution. We performed a comprehensive analysis of SDRs and SSRs across the complete plastomes of 79 \u003cem\u003eIris\u003c/em\u003e species. Four types of SDRs\u0026mdash;Forward, Reverse, Palindromic, and Complementary repeats\u0026mdash;were identified using the online tool REPuter [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bibiserv.cebitec.uni-bielefeld.de/reputer\u003c/span\u003e\u003cspan address=\"https://bibiserv.cebitec.uni-bielefeld.de/reputer\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The parameters were set as follows: Hamming distance\u0026thinsp;=\u0026thinsp;3, maximum number of repeats\u0026thinsp;=\u0026thinsp;500, and minimum repeat length\u0026thinsp;=\u0026thinsp;30 bp. Additionally, SSRs were detected using the MISA tool [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] (MISA-web, IPK Gatersleben). Six categories of microsatellites were searched, i.e., Mono-nucleotide, Di-nucleotide, Tri-nucleotide, Tetra-nucleotide, Penta-nucleotide, and Hexa-nucleotide repeats. The minimum repeat thresholds were set at 10, 5, 4, 3, 3, and 3, respectively [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e1.4 Selective pressure analysis\u003c/h2\u003e\u003cp\u003eThe selective pressure on protein-coding genes (PCGs) of \u003cem\u003eIris\u003c/em\u003e plastomes was assessed based on the variation in nucleotide substitution rates [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Coding sequences (CDSs) were extracted, and the ratios of nonsynonymous (dN) to synonymous (dS) substitution rates (dN/dS) were estimated using the CODEML module implemented in PAML v4.9 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. A constraint tree generated by a genetic algorithm based on the CDS dataset was used as the input phylogeny. The parameters in the CODEML control file were set as follows: (1) F3 \u0026times; 4 model for codon frequencies; (2) \u0026ldquo;model\u0026thinsp;=\u0026thinsp;0\u0026rdquo; for allowing a single dN/dS value to vary among branches; (3) \u0026ldquo;cleandata\u0026thinsp;=\u0026thinsp;1\u0026rdquo; to remove gaps; and (4) default settings for other parameters [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This analysis provided robust support for estimating lineage-specific selective constraints acting on plastid PCGs in \u003cem\u003eIris\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e1.5 Comparative plastome alignment and sequence divergence analysis\u003c/h2\u003e\u003cp\u003eTo evaluate the sequence divergence and the hypervariable regions among \u003cem\u003eIris\u003c/em\u003e species, we conducted global plastome alignments of 36 \u003cem\u003eIris\u003c/em\u003e species using the mVISTA software [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] in Shuffle-LAGAN alignment mode, with \u003cem\u003eIris germanica\u003c/em\u003e (GenBank accession: NC_062594) serving as the reference genome. This analysis revealed patterns of sequence variation and facilitated the identification of highly variable loci that may serve as potential molecular markers for species delimitation and phylogenetic reconstruction of closely related taxa. To more precisely locate the genomic regions with high mutation rates, we selected the 79 \u003cem\u003eIris\u003c/em\u003e plastomes to perform sliding window analysis using the Genepioneer platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cloud.genepioneer.com\u003c/span\u003e\u003cspan address=\"http://cloud.genepioneer.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and DnaSP v6.12.03 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Prior to the analysis, multiple sequence alignment of plastid genomes was carried out using MAFFT v7.505 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] with default parameters. The sliding window analysis was conducted with a window length of 400 bp and a step size of 200 bp. Nucleotide diversity (Pi) values were calculated for each window and plotted to visualize the distribution of sequence variation across the plastomes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e1.6 Phylogenetic analysis\u003c/h2\u003e\u003cp\u003eTo explore the phylogenetic relationships among \u003cem\u003eIris\u003c/em\u003e species, we analyzed both complete plastome and coding sequence (CDS) datasets of 79 \u003cem\u003eIris\u003c/em\u003e species, using \u003cem\u003eC. cartwrightianus\u003c/em\u003e and \u003cem\u003eC. sativus\u003c/em\u003e as outgroups. Sequence alignment was performed using MAFFT v7.505 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], followed by manual inspection and adjustment. Non-conserved regions were trimmed using trimAl v1.2 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Phylogenetic trees were reconstructed using three methods: maximum likelihood (ML) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] in IQ-TREE v2.4.0 [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], maximum parsimony (MP) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] in MEGA11 [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], and Bayesian inference (BI) in MrBayes v3.2.6 [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. For ML analyses, the best-fit model (TVM\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;R4) was selected by ModelFinder [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] for both datasets. For BI, the best-fit model (GTR\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G) was identified using Modeltest v3.7 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Markov chain Monte Carlo (MCMC) analysis was run for 50\u0026nbsp;million generations with four chains, sampling every 1,000 generations and discarding the 25% as burn-in. Support values for both the complete plastome and CDS datasets were assessed using 1,000 ultrafast bootstrap replicates [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The generated phylogenetic trees were visualized using FigTree v1.4.4 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] and iTOL [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and further beautified in Adobe Illustrator 2024 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e1.7 Molecular clock dating\u003c/h2\u003e\u003cp\u003eThe estimates of divergence times were based on the molecular clock theory, which assumes that codon substitutions in gene sequences occur at a nearly constant rate [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], and genetic differences between species are positively correlated with divergence times [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. We used the plastomes of 79 \u003cem\u003eIris\u003c/em\u003e species for divergence time estimation, with a species tree constructed from complete plastome sequences as input. Sequences were aligned using MAFFT v. 7.526 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], and the alignments were manually checked and adjusted to ensure accuracy. Non-conserved regions were trimmed using trimAl v. 1.2 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Phylogenetic trees were inferred using IQ-TREE v. 2.4.0 [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], with the CDS and complete plastome datasets modeled under TVM\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;R4 as selected by ModelFinder [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Node support was evaluated by 1,000 rapid bootstrap replicates [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Divergence time calibration among species in the phylogenetic tree was based on fossil and molecular data integrated from the TimeTree database [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://timetree.org/\u003c/span\u003e\u003cspan address=\"http://timetree.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The estimated divergence time range was set within \u0026plusmn;\u0026thinsp;0.04 Ma of the fossil- and molecular-based estimates. Fossil calibration points included the most recent common ancestor of \u003cem\u003eCrocus\u003c/em\u003e and \u003cem\u003eIris\u003c/em\u003e, as well as root nodes within \u003cem\u003eIris\u003c/em\u003e species pairs such as \u003cem\u003eIris domestica\u003c/em\u003e and \u003cem\u003eIris dichotoma\u003c/em\u003e, \u003cem\u003eIris sanguinea\u003c/em\u003e and \u003cem\u003eIris pseudacorus\u003c/em\u003e, \u003cem\u003eIris odaesanensis\u003c/em\u003e and \u003cem\u003eIris minutoaurea\u003c/em\u003e, and \u003cem\u003eIris decora\u003c/em\u003e and \u003cem\u003eIris. japonica\u003c/em\u003e, resulting in a fossil-calibrated rooted phylogeny. Using this phylogeny, divergence times were estimated with the MCMCTree [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] method implemented in PAML 4.9 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Markov chain Monte Carlo (MCMC) analyses were run for 100\u0026nbsp;million generations with four independent chains, sampling every 1,000 generations and discarding the first 25% as burn-in. Convergence of the MCMC runs was assessed using Tracer v.1.7.2 [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The resulting phylogeny was visualized with Figtree v.1.4.4 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] and the online tool TVBOT [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chiplot.online/tvbot.htm\u003c/span\u003e\u003cspan address=\"https://chiplot.online/tvbot.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Plastome features\u003c/h2\u003e\u003cp\u003eWe analyzed the structure of 79 complete plastomes of \u003cem\u003eIris\u003c/em\u003e species, including 17 newly sequenced plastomes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The plastome lengths ranged from 150,169 bp to 155,878 bp. All plastomes exhibited a typical quadripartite structure, consisting of a large single-copy (LSC) region, a small single-copy (SSC) region, and two inverted repeats (IRa and IRb) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The overall GC content was highly conserved among all species, ranging from 37.32% to 38.23% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Each plastome was annotated and manually checked, yielding 123 to 132 genes per plastome, including 84 to 88 protein-coding genes, 34 to 41 transfer RNA (tRNA) genes, and 0 to 8 ribosomal RNA (rRNA) genes (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Taking \u003cem\u003eIris decora\u003c/em\u003e as an example, its plastome contains 132 genes, of which 76 are involved in self-replication, 45 are associated with photosynthesis, and 11 are related to other functions. Among these, 20 genes are present in two copies (located in the IR regions), while the remaining 112 genes are present in a single copy. Further analysis revealed that 13 genes contain a single intron, 3 genes contain two introns, and the remaining 116 genes lack introns (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBasic characteristics of the plastomes generated in this study.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"14\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSize\u003c/p\u003e\u003cp\u003e(bp)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e\u003cp\u003eGC length (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLSC\u003c/p\u003e\u003cp\u003elength(bp)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSSC\u003c/p\u003e\u003cp\u003elength(bp)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eIR\u003c/p\u003e\u003cp\u003elength(bp)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003cp\u003enumber\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eProtein-coding gene number\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003erRNA\u003c/p\u003e\u003cp\u003egene\u003c/p\u003e\u003cp\u003enumber\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c14\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003etRNA\u003c/p\u003e\u003cp\u003egene\u003c/p\u003e\u003cp\u003enumber\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCoding\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLSC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSSC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eIR\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris barbatula\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e153600\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e35.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e43.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e82791\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e18467\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e26171\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e127\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris bulleyana\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e152198\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e38.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e36.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e43.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e82085\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e18003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e26055\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e127\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris cangshanensis\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e154008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e35.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e30.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e42.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e83559\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e17937\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e26256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris chrysographes\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e153570\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e38.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e36.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e42.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e81688\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e16991\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e27445\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris collettii\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e154165\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e35.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e42.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e83120\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e18567\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e26239\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e126\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris decora\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e153787\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e35.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e43.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e82973\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e18470\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e26172\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e132\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris delavayi\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e152397\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e38.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e36.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e43.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e82319\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e18008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e26035\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e127\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris latistyla\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e155878\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e36.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e42.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e81323\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e18513\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e28021\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris leptophylla\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e155138\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e35.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e42.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e81761\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e18455\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e27461\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris milesii\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e153202\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e36.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e42.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e82778\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e18536\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e25944\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e131\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris pumila\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e153546\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e35.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e43.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e82655\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e18457\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e26217\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris reticulata\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e154636\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e36.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e42.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e82787\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e15807\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e28021\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e127\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris scariosa\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e154814\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e36.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e42.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e81888\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e16022\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e28452\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris tectorum\u003c/b\u003e \u003cb\u003ef.\u003c/b\u003e \u003cb\u003ealba\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e153199\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e36.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e42.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e82779\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e18562\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e25929\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e131\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris tenuifolia\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e150487\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e38.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e36.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e43.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e81136\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e18035\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e25658\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris ventricosa\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e150169\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e38.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e36.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e32.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e42.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e84511\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e17302\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e25663\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e123\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIris wattii\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e153867\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e36.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e42.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e82298\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e18515\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e26527\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e132\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.2 IR boundary comparative analysis\u003c/h2\u003e\u003cp\u003eWe analyzed four junctions of 79 \u003cem\u003eIris\u003c/em\u003e plastomes (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), i.e., JLB (junction between LSC and IRb), JSB (between SSC and IRb), JSA (between SSC and IRa), and JLA (between LSC and IRa). At the JLB junction, most species exhibited boundaries between \u003cem\u003erpl22\u0026ndash;trnH\u003c/em\u003e and \u003cem\u003erpl22\u0026ndash;rps19\u003c/em\u003e. \u003cem\u003eIris leptophylla\u003c/em\u003e and \u003cem\u003eIris latistyla\u003c/em\u003e located the JLB junction in \u003cem\u003erpl16\u003c/em\u003e. \u003cem\u003eIris chrysographes\u003c/em\u003e and \u003cem\u003eIris scarios\u003c/em\u003e lied the JLB boundary between \u003cem\u003erps3\u003c/em\u003e and \u003cem\u003erpl22\u003c/em\u003e. \u003cem\u003eIris confusa\u003c/em\u003e, \u003cem\u003eIris luojiensis\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and \u003cem\u003eI. japonica\u003c/em\u003e fall their JLB boundaries in \u003cem\u003erps19\u003c/em\u003e. At the JSB junction, most boundaries were located within \u003cem\u003eycf1\u003c/em\u003e or \u003cem\u003endhF\u003c/em\u003e, while \u003cem\u003eIris mariae\u003c/em\u003e lied its JSB boundary between \u003cem\u003erps7\u003c/em\u003e and \u003cem\u003etrnV\u003c/em\u003e. \u003cem\u003eYcf1\u003c/em\u003e was contracted into the IRb region in \u003cem\u003eIris dichotoma\u003c/em\u003e and \u003cem\u003eIris japonica\u003c/em\u003e. \u003cem\u003eNdhF\u003c/em\u003e did not extend into the IRb region in \u003cem\u003eIris cangshanensis\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. At the JSA junction, the boundary was commonly located within \u003cem\u003eycf1\u003c/em\u003e. However, JSA junction lied between \u003cem\u003etrnV\u0026ndash;rps7\u003c/em\u003e in \u003cem\u003eIris mariae\u003c/em\u003e, and lied within \u003cem\u003endhF\u003c/em\u003e in \u003cem\u003eIris ventricosa\u003c/em\u003e and \u003cem\u003eIris missouriensis\u003c/em\u003e. Moreover, \u003cem\u003eycf1\u003c/em\u003e in \u003cem\u003eIris missouriensis\u003c/em\u003e was contracted into the IRa region. At the JLA junction, the junction typically lied between \u003cem\u003etrnH\u0026ndash;psbA\u003c/em\u003e or \u003cem\u003erps19\u0026ndash;psbA\u003c/em\u003e. In \u003cem\u003eIris wattii\u003c/em\u003e, the junction was located within \u003cem\u003epsbA\u003c/em\u003e. Gene content analysis revealed that 45 species contained two copies of \u003cem\u003eycf1\u003c/em\u003e, 33 species only had a single copy of \u003cem\u003eycf1\u003c/em\u003e, and one species lacked \u003cem\u003eycf1\u003c/em\u003e entirely. All species except \u003cem\u003eIris mariae\u003c/em\u003e contained the \u003cem\u003endhF\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Codon usage preference\u003c/h2\u003e\u003cp\u003eCodon counts of 79 \u003cem\u003eIris\u003c/em\u003e plastomes ranged from 27,149 in \u003cem\u003eIris goniocarpa\u003c/em\u003e to 29,405 in \u003cem\u003eIris scariosa\u003c/em\u003e (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Codon usage frequencies were largely conserved across species (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Among the 20 amino acids encoded by 64 codons, only methionine (Met) and tryptophan (Trp) were represented by a single codon (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The relative synonymous codon usage (RSCU) values across all species showed low overall variation (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). The three codons with the highest RSCU values were CGA (Arg), CUG (Leu), and GUU (Val), and the lowest values were UUG (Leu), UUA (Leu), and AGG (Arg) (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e, Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). A histogram of CBI values across \u003cem\u003eIris\u003c/em\u003e species revealed a narrow distribution, with the majority of genes exhibiting values tightly clustered between \u0026minus;\u0026thinsp;0.112 and \u0026minus;\u0026thinsp;0.109. This indicates a consistent pattern of mild codon usage bias across genes. No genes showed positive CBI values, suggesting an overall absence of strong preference for optimal codons in the examined dataset (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Further analysis revealed that codons ending in T or A generally had RSCU values\u0026thinsp;\u0026gt;\u0026thinsp;1, while those ending in C or G typically had RSCU values\u0026thinsp;\u0026lt;\u0026thinsp;1 (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). These findings suggest a bias toward the use of T and A at the third codon position [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], a pattern commonly observed in angiosperm plastid genomes [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Moreover, GC content analysis showed that GC2 values were significantly higher than GC3 values across all \u003cem\u003eIris\u003c/em\u003e species, indicating a stronger preference for G/C bases at the second codon position (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Repeat analysis\u003c/h2\u003e\u003cp\u003eThe number of SDRs varied markedly among species, ranging from 28 in \u003cem\u003eIris speculatrix\u003c/em\u003e to 200 in \u003cem\u003eIris cangshanensis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Classification of repeat types revealed that palindromic repeats (~\u0026thinsp;48.0%) and forward repeats (~\u0026thinsp;42.4%) were the most, while reverse (~\u0026thinsp;7.5%) and complement repeats (~\u0026thinsp;2.1%) were relatively rare (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). For the SSRs, the number detected per plastome ranged from 31 in \u003cem\u003eIris halophila\u003c/em\u003e to 74 in \u003cem\u003eIris germanica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). Among the identified SSR types, Mono-nucleotide repeats were the most abundant (~\u0026thinsp;60.2%), followed by Di-nucleotide repeats (~\u0026thinsp;21.2%). Hexa-nucleotide repeats were the least frequent, accounting for only\u0026thinsp;~\u0026thinsp;0.5% of the total SSRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Selective pressure analysis\u003c/h2\u003e\u003cp\u003eThe dN/dS ratio is a widely used indicator of selection: dN/dS\u0026thinsp;=\u0026thinsp;1 suggest neutral evolution, dN/dS\u0026thinsp;\u0026lt;\u0026thinsp;1 indicate purifying (negative) selection, and dN/dS\u0026thinsp;\u0026gt;\u0026thinsp;1 imply diversifying (positive) selection. Across the genes analyzed, dN values ranged from 0 to 0.0430, and dS values from 0.0062 to 0.1239, with corresponding dN/dS ratios spanning 0 to 0.9215 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). \u003cem\u003ecemA\u003c/em\u003e exhibited the highest nonsynonymous substitution rate (dN\u0026thinsp;=\u0026thinsp;0.0430), while \u003cem\u003epetD\u003c/em\u003e had the highest synonymous rate (dS\u0026thinsp;=\u0026thinsp;0.1239). Notably, \u003cem\u003eycf2\u003c/em\u003e showed the highest dN/dS ratio (0.9215) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). In contrast, \u003cem\u003epsbT\u003c/em\u003e (dN/dS\u0026thinsp;=\u0026thinsp;0), \u003cem\u003epsbD\u003c/em\u003e (dN/dS\u0026thinsp;=\u0026thinsp;0.0033), and \u003cem\u003eatpH\u003c/em\u003e (dN/dS\u0026thinsp;=\u0026thinsp;0.0033) exhibited the lowest dN/dS values (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Comparative plastome alignment and detection of hypervariable regions\u003c/h2\u003e\u003cp\u003eThe global alignment of 36 representative taxa revealed pronounced sequence divergence in both coding and non-coding regions. Several protein-coding genes\u0026mdash;\u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003erps16\u003c/em\u003e, and \u003cem\u003endhF\u003c/em\u003e\u0026mdash;exhibited substantial variation. Additionally, intergenic regions such as \u003cem\u003etrnS-trnG\u003c/em\u003e, \u003cem\u003etrnD-trnY\u003c/em\u003e, \u003cem\u003epetA-psbJ\u003c/em\u003e, \u003cem\u003endhF-rpl32\u003c/em\u003e, \u003cem\u003erps7-trnV\u003c/em\u003e, \u003cem\u003erpl22-rps19\u003c/em\u003e, and \u003cem\u003erbcL-accD\u003c/em\u003e showed high sequence divergence (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA, B). Pi values across the plastomes of 79 \u003cem\u003eIris\u003c/em\u003e species ranged from 0.00043 to 0.06705, with an average nucleotide diversity of 0.019. Comparative analysis of genome structure revealed that the SSC region exhibited significantly higher genetic variation than LSC and IR regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). \u003cem\u003eYcf1\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.06705, 0.06510), \u003cem\u003endhF\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.06018), and intergenic spacer \u003cem\u003eccsA-psaC\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.04704) harbored the highest nucleotide variation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results underscored the SSC as the most variable plastome region across \u003cem\u003eIris\u003c/em\u003e species. In addition to these hypervariable loci in the SSC, several regions within the LSC also exhibited elevated levels of genetic diversity, including the intergenic region \u003cem\u003etrnS-trnG\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.04420), the coding gene \u003cem\u003erpl16\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.03921), and the spacer \u003cem\u003epetA-psbJ\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.03572) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Table\u0026nbsp;2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eTable\u0026nbsp;2 Seven hypervariable region genes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGenes\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePi (Nucleotide diversity)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eS (Number of polymorphic sites)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePosition\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eycf1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.06705\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e264\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSSC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eycf1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.06510\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e277\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSSC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003endhF\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.06018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e244\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSSC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eccsA-ndhD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.04704\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e264\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSSC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003etrnS-trnG\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.04420\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e190\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLSC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003erpl16\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.03921\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLSC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003epetA-psbJ\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.03572\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e139\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLSC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Phylogenetic relationships\u003c/h2\u003e\u003cp\u003eBoth Maximum Likelihood (ML), Maximum Parsimony (MP), and Bayesian Inference (BI) analyses produced highly congruent phylogenetic trees, supporting an almost identical overall topology (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e; Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Both CDS and complete plastome datasets yielded largely concordant topologies, with only two inconsistencies observed (Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Overall, the complete plastome dataset provided better phylogenetic resolution than the CDS dataset, as indicated by higher bootstrap support values (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e; Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Of particular note, \u003cem\u003eIris luojiensis\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], a species recently described in June 2024, was clustered with subgen. \u003cem\u003eCrossiris\u003c/em\u003e across all phylogenetic inference methods. It received strong nodal support (ML\u0026thinsp;=\u0026thinsp;99, MP\u0026thinsp;=\u0026thinsp;98, BI\u0026thinsp;=\u0026thinsp;1.00), highlighting the robustness and taxonomic stability of its phylogenetic placement (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e; Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). In contrast, \u003cem\u003eIris cangshanensis\u003c/em\u003e [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] was resolved as a strongly supported monophyletic lineage, receiving full support from all three analytical frameworks (ML\u0026thinsp;=\u0026thinsp;100, MP\u0026thinsp;=\u0026thinsp;100, BI\u0026thinsp;=\u0026thinsp;1.00). This finding highlights its substantial phylogenetic divergence from other \u003cem\u003eIris\u003c/em\u003e species and supports its recognition as a distinct evolutionary lineage (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e; Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). The phylogenetic positions of species in other lineages remained stable, and the tree constructed using the complete plastome dataset exhibited significantly stronger support than that based on CDSs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e; Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). A comparative analysis was conducted on 79 \u003cem\u003eIris\u003c/em\u003e species focusing on two morphological traits: the type of appendages on the falls and the form of underground storage organs, indicating the original traits of \u003cem\u003eIris\u003c/em\u003e being bearded and rhizomelic (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Divergence time estimation\u003c/h2\u003e\u003cp\u003eThe initial divergence between \u003cem\u003eIris\u003c/em\u003e and \u003cem\u003eCrocus\u003c/em\u003e was estimated at approximately 49.00\u0026nbsp;million years ago (Ma) during the Eocene epoch, with a 95% highest posterior density (HPD) interval of 44.55\u0026ndash;52.41 Ma (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Within subgen. \u003cem\u003eCrossiris\u003c/em\u003e, multiple lineage divergences occurred during the Miocene to Pliocene epochs, with estimated dates at 21.43 Ma (95% HPD: 16.02\u0026ndash;27.54 Ma), 4.31 Ma (1.77\u0026ndash;7.16 Ma), 4.21 Ma (1.63\u0026ndash;7.19 Ma), and 2.82 Ma (0.63\u0026ndash;5.63 Ma). Divergence within subgen. \u003cem\u003eIris\u003c/em\u003e was estimated at ~\u0026thinsp;10.37 Ma (95% HPD: 5.47\u0026ndash;20.00 Ma, Miocene), with a much more recent event at 0.02 Ma (95% HPD: 0\u0026ndash;0.08 Ma, Quaternary), indicating the coexistence of both ancient and recently evolved lineages. Major divergence events in subgen. \u003cem\u003eNepalensis\u003c/em\u003e were dated to 13.56 Ma (95% HPD: 8.43\u0026ndash;18.93 Ma, Miocene) and 2.41 Ma (95% HPD: 0.36\u0026ndash;4.68 Ma, Quaternary), suggesting substantial lineage diversification across geological timescales. Additionally, Subgen. \u003cem\u003ePardanthopsis\u003c/em\u003e and Subgen. \u003cem\u003eHermodactyloides\u003c/em\u003e exhibited more recent divergence times of 0.40 Ma (95% HPD: 0.21\u0026ndash;0.62 Ma) and 0.24 Ma (95% HPD: 0.05\u0026ndash;0.45 Ma), respectively. The primary divergence event of Subgen. \u003cem\u003eScorpiris\u003c/em\u003e could date back to 6.66 Ma (95% HPD: 0.37\u0026ndash;9.77 Ma) within Miocene epoch (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). For recently described species, \u003cem\u003eIris cangshanensis\u003c/em\u003e [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] was estimated to have originated approximately 44.40 Ma (95% HPD: 37.03\u0026ndash;51.25 Ma, Eocene), while \u003cem\u003eIris luojiensis\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] diverged at around 3.65 Ma (95% HPD: 1.24\u0026ndash;6.33 Ma, Pliocene). Overall, divergence times among major \u003cem\u003eIris\u003c/em\u003e species ranged from 0.02 to 25.00 Ma, primarily within the Miocene to Quaternary periods, indicating multiple episodes of lineage radiation and ecological adaptation throughout the Cenozoic.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cb\u003ePlastome characteristics of\u003c/b\u003e \u003cb\u003eIris\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe plastome structure across 79 \u003cem\u003eIris\u003c/em\u003e spcecies is largely conserved. All plastomes exhibit typical quadripartite structure, consisting of a large single-copy (LSC) region, a small single-copy (SSC) region, and two inverted repeats (IRa and IRb) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Such structure is similar to that reported among other studies on \u003cem\u003eIris\u003c/em\u003e [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Similar to most angiosperm plastomes, the conservation of plastome structure is likely consistent to the requirement of maintaining the stability of plastome functionality [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e123 to 132 genes are detected per plastome of \u003cem\u003eIris\u003c/em\u003e, including 84 to 88 protein-coding genes. 76 among these genes are involved in self-replication, 45 are associated with photosynthesis, and 11 are related to other functions. Expansions and contractions are observed in the IR regions of the 79 \u003cem\u003eIris\u003c/em\u003e plastomes. IR regions of angiosperm plastomes generally begin near rps19 gene and end consistently downstream at trnN-GUU or ycf1 gene [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. While IR expansion has been documented in specific lineages, usually within the LSC region [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. We discover four junctions: JLB (junction between LSC and IRb), JSB (between SSC and IRb), JSA (between SSC and IRa), and JLA (between LSC and IRa) in this study. These findings suggest that IR expansions are independent events in the genus \u003cem\u003eIris\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eAn analysis of different SSR repeat types reveals that mononucleotide repeats are the most prevalent (~\u0026thinsp;60.2%), followed by Di-nucleotide repeats (~\u0026thinsp;21.2%). SSR numbers varied slightly across the 79 \u003cem\u003eIris\u003c/em\u003e taxa, ranging from 31 to 74. The predominance of palindromic and forward repeats suggests their potential roles in maintaining plastome structural stability and facilitating intragenomic recombination. This study aligns with other plastome studies of \u003cem\u003eIris\u003c/em\u003e [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Together, these results characterize the distribution patterns of repetitive elements in \u003cem\u003eIris\u003c/em\u003e plastid genomes and provide a valuable basis for future evolutionary studies and the development of plastid-based molecular markers.\u003c/p\u003e\u003cp\u003eFactors influencing codon usage include genome size, base mutation, genetic drift, natural selection, gene expression level, and protein structure [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Synonymous codons arise from mutations and may differ in their usage frequency, which can be quantified by relative synonymous codon usage (RSCU), a measure that reveals codon usage preferences among genes [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Codon counts of 79 \u003cem\u003eIris\u003c/em\u003e plastomes range from 27,149 in \u003cem\u003eIris goniocarpa\u003c/em\u003e to 29,405 in \u003cem\u003eIris scariosa\u003c/em\u003e (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Codon usage frequencies are largely conserved across species (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Among the 20 amino acids encoded by 64 codons, only methionine (Met) and tryptophan (Trp) are represented by a single codon (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e65 genes are identified to undergo negative selection with the corresponding dN/dS ratios ranging from 0 to 0.9215. Most of the negative selective genes are involved in self-replication, photosynthesis, and protein synthesis. Among them, ycf2 shows the highest dN/dS ratio (0.9215), suggesting it undergoes relaxed purifying selection pressure with elevated evolutionary rate, potentially due to relaxed functional constraints or lineage-specific adaptive evolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). In contrast, psbT (dN/dS\u0026thinsp;=\u0026thinsp;0), psbD (dN/dS\u0026thinsp;=\u0026thinsp;0.0033), and atpH (dN/dS\u0026thinsp;=\u0026thinsp;0.0033) exhibited the lowest dN/dS values, indicating they undergo strong purifying selection with high evolutionary conservation. These negative selected genes are known to play critical roles in photosynthetic complexes, which may account for their functional constraint (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). Collectively, these findings highlight the heterogeneity of selective pressures across \u003cem\u003eIris\u003c/em\u003e plastid genes and provide molecular insights into the evolutionary history and adaptive divergence of genus \u003cem\u003eIris\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic analysis and adaptive evolution\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCompared to previous phylogenetic studies on \u003cem\u003eIris\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], this study is the most comprehensive one to analyze \u003cem\u003eIris\u003c/em\u003e phylogenetic relationships with density sampling using plastome-scale sequences. Our plastome tree provides a more robust phylogenetic framework for the genus \u003cem\u003eIris\u003c/em\u003e compared to the studies based on single or multiple locus DNA sequences, with major nodes showing strong support (i.e., PP\u0026thinsp;=\u0026thinsp;1.00 and BS\u0026thinsp;\u0026ge;\u0026thinsp;80). The results further confirmed that whole plastome sequencing can enhance the phylogenetic resolution within a given lineage. Most \u003cem\u003eIris\u003c/em\u003e species were resolved as clustered into a monophyletic based on the plastome sequence data, except for \u003cem\u003eIris cangshanensis\u003c/em\u003e, which is inferred to be a monophyletic lineage that locates at the basal of the \u003cem\u003eIris\u003c/em\u003e clade. It implies that \u003cem\u003eIris cangshanensis\u003c/em\u003e may have a more complex taxonomic relationship and evolutionary history than we previously thought [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study is the first to analyze the maternal evolutionary history of the genus \u003cem\u003eIirs\u003c/em\u003e based on a large dataset of plastomes. Notably, our plastome-based phylogenomic tree indicates three major clades in \u003cem\u003eIris\u003c/em\u003e with high credibility support (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The clade A composes of subgenus \u003cem\u003eIris\u003c/em\u003e and subgenus \u003cem\u003ePardanthopsis\u003c/em\u003e. The clade B includes subgenus \u003cem\u003eScorpiris\u003c/em\u003e, subgenus \u003cem\u003eCrossiris\u003c/em\u003e and subgenus \u003cem\u003eNepalensis\u003c/em\u003e. The clade C composes of subgenus \u003cem\u003eLimmirs\u003c/em\u003e, \u003cem\u003eIris scariosa\u003c/em\u003e, \u003cem\u003eIris reticulate\u003c/em\u003e, \u003cem\u003eIris halophila\u003c/em\u003e and \u003cem\u003eIris speculatrix\u003c/em\u003e. This result is partially consistent with the previous phylogeny tree based on 19 \u003cem\u003eIris\u003c/em\u003e plastomes [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. As for the previous phylogeny studies using cpDNA sequences on \u003cem\u003eIris\u003c/em\u003e by Wilson [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and Guo et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], some of their results are also consistent with this study. For examples, Wilson's findings indicate that the subgenus \u003cem\u003eLimmirs\u003c/em\u003e is an independent branch, while Guo's results suggest that subgenus \u003cem\u003eScorpiris\u003c/em\u003e, subgenus \u003cem\u003eCrossiris\u003c/em\u003e and subgenus \u003cem\u003eNepalensis\u003c/em\u003e form a clade.\u003c/p\u003e\u003cp\u003eHybridization and introgression commonly occur among \u003cem\u003eIris\u003c/em\u003e with sympatric distributions [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. When interspecific gene flow is asymmetric, one parental species may experience assimilation of its nuclear genome, while its maternal plastome is retained in the populations. This phenomenon is commonly referred to as the introgression-induced chloroplast capture [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Introgression-induced chloroplast capture has been identified as a mechanism that can distort phylogenetic relationships, often resulting in geographic clustering of introgressed taxa. Natural hybridization and introgression are commonly observed among the species within \u003cem\u003eIris\u003c/em\u003e [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. The present phylogenetic work on \u003cem\u003eIris\u003c/em\u003e revealed a secondary increase in the speciation rate of \u003cem\u003eIris\u003c/em\u003e during Oligocene and Miocene, suggesting that interspecific hybridization may have occurred during the early stages of its diversification (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Incomplete lineage sorting among taxa is often associated with radiations [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Accordingly, the possibility of incomplete lineage sorting causing cytonuclear discordance cannot be totally discounted. Furthermore, pollen and seed dispersal are critical determinants of gene flow [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Gene flow via pollen is significantly greater than that occurring via seeds, leading to broader genetic exchange for the nuclear genome compared to the plastome. Differences between seed- and pollen-mediated gene flow can result in cytonuclear discordance in phylogenetic studies [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. \u003cem\u003eIris\u003c/em\u003e species are primarily pollinated by insects in natural habitats [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e], thus achieve the long-distance transmission possibility of pollen enhances gene flow among populations. In contrast, the seed dispersal of \u003cem\u003eIris\u003c/em\u003e is more limited [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. The contrasting patterns of pollen- and seed-mediated gene flow among the ancestral populations could contribute to the cyto-nuclear discordance observed in \u003cem\u003eIris\u003c/em\u003e [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. We found significantly nuclear-cytoplasmic conflict of genus \u003cem\u003eIris\u003c/em\u003e (unpublished data), but all the hypotheses above should be tested concisely in future studies.\u003c/p\u003e\u003cp\u003eCorrect phylogeny and divergence-time estimation are essential for evolutionary history study. This study is the first to conduct divergence-time estimation on \u003cem\u003eIris\u003c/em\u003e. An appropriate molecular markers selected is of great concern when inferring a phylogeny of targeted taxa, as the selected markers can strongly affect overall topology and divergence time estimates. With a complete chloroplast gene set, we can choose suitable genes to facilitate and optimize divergence-time estimation. The present divergence-time estimation indicates that the crown node age of \u003cem\u003eIirs\u003c/em\u003e is estimated at 49.00\u0026nbsp;million years ago (Ma). \u003cem\u003eIris cangshanensis\u003c/em\u003e diversifed from major \u003cem\u003eIris\u003c/em\u003e clade at 44.40 Ma. The differentiation of subgenus \u003cem\u003eLimniris\u003c/em\u003e clade between other groups of the \u003cem\u003eIris\u003c/em\u003e occurred at Oligocene (29.82 Ma), and the clade B radiated at Miocene (20.19 Ma) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study conducts certain investigations into the morphological evolution of \u003cem\u003eIris\u003c/em\u003e genus. We mainly focus on two key traits of \u003cem\u003eIris\u003c/em\u003e, i.e., the morphology of the underground organs and the form of the nectary guide. We classify the nectar guide on the falls into three types, i.e., beards, crests, and those with spots, and divide the root morphology into tuberous, bulbous and rhizomelic (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The present results indicate that the beard or crest on falls occurred independently multiple times in subgen. \u003cem\u003eIris\u003c/em\u003e, subgen. \u003cem\u003eNepalensis\u003c/em\u003e, and subgen. \u003cem\u003eCrossiris\u003c/em\u003e, respectively. As for the root morphology evolution in \u003cem\u003eIris\u003c/em\u003e, the present results indicate that rhizome might be the original trait of \u003cem\u003eIris\u003c/em\u003e and independently evolved into bulbous (Subgenus \u003cem\u003eScorpiris\u003c/em\u003e) or tuberous (Subgenus \u003cem\u003eNepalensis\u003c/em\u003e). The plastome-based phylogenetic framework does not align with key taxonomical groupings based on the underground organ or the appendage on fall (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), indicating that these morphological traits are limited phylogenetically informative at the subgenera ranks in some subgenera classification of the genus \u003cem\u003eIris\u003c/em\u003e. The paraphyletic pattern of beard species on the plastome trees may indicate a consequence of convergent adaptation to cope with animal predation [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e], and the independently origin of the bulbous or tuberous root may indicate the adaptation for the special external inorganic environment [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we sequence and assemble the complete chloroplast genomes of 17 \u003cem\u003eIris\u003c/em\u003e specie, and compare the structure of 79 \u003cem\u003eIris\u003c/em\u003e plastomes by adding 62 published samples. Phylogenetic analysis based on the chloroplast genome supported part of the previous subgenera taxonomic treatment study using morphological characteristics and fragments sequences. Divergence time analysis revealed that \u003cem\u003eIris\u003c/em\u003e originated at early Eocene and diversified at early Oligoceae. Beard or crest species, in combination with the rhizomelic roots, might be the original traits of genus \u003cem\u003eIris\u003c/em\u003e, and independently evolve into beardless, tuberous and bulbous. Overall, this study demonstrates that the whole chloroplast genome sequences display variable information to resolve phylogenetic relationships in this genus.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eThe collecting of all samples in this study followed the Regulations on the Protection of Wild Plants of China, 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. All methods were carried out in accordance with relevant guidelines and regulations.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe present research was funded by the National Natural Science Foundation of China (Grant No. 32460335), the Foundation of Yunnan Province Science and Technology Department (Grant No. 202305AM070003), and the National Natural Science Foundation of China (32100169).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJinfeng Liu, Xin Jin and Xianfeng Jiang collected samples, Jinfeng Liu and Xingtang Du were responsible for the overall analysis of the data of the article, Yanping Xie provided comprehensive guidance for the overall analysis, and Xianfeng Jiang was responsible for the writing and revision of the article.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data presented in the study are deposited in the GenBank repository (https://www.ncbi.nlm.nih.gov/genbank/), and the accession numbers showed in Table S1.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBritish Iris Society. 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Ann Botany. 2009;103(5):687\u0026ndash;702.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Epilepsy, Stigma, Depression, Quality of life, Rural","lastPublishedDoi":"10.21203/rs.3.rs-7909828/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7909828/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e \u003cem\u003eIris\u003c/em\u003e is a group of perennial herb with important horticultural value. There are over 300 species of \u003cem\u003eIris\u003c/em\u003e species worldwide, and China has approximately 70 species. A series of studies utilized chloroplast fragments to explore the phylogenetic relationships of Irises, but the number of species and the reliability of the results of most studies were not satisfied. We sequenced and assembled the chloroplast genomes of 17 species of \u003cem\u003eIris\u003c/em\u003e, and downloaded 62 available data from public database to construct the most complete phylogenetic evolutionary tree and analyze the evolution and origin relationship of genus \u003cem\u003eIris.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e The 79 \u003cem\u003eIris\u003c/em\u003e chloroplast genomes exhibit highly similar genome size, gene content, and order. The \u003cem\u003eIris\u003c/em\u003e chloroplast genomes show typical quadripartite structures with lengths from 150,169 bp to 155,878 bp. All plastomes exhibit typical quadripartite structure, consisting of a large single-copy (LSC) region, a small single-copy (SSC) region, and two inverted repeats (IRa and IRb). Phylogenetic results support \u003cem\u003eIris\u003c/em\u003e as a monophyletic group, and indicate that \u003cem\u003eIris\u003c/em\u003e was divided into three major clades. The divergence times indicate that \u003cem\u003eIris\u003c/em\u003e diverged from \u003cem\u003eCrocos\u003c/em\u003e at early Eocenein. Bearded and rhizomelic might be the original traits of \u003cem\u003eIris\u003c/em\u003e, and then independently evolve into beardless, bulbous or tuberous.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e Our study supports the present taxonomic treatment at the subgenus level for \u003cem\u003eIris\u003c/em\u003e species, and demonstrates the validity of phylogenetic resolution using whole chloroplast genome sequences. We also prove that the \u003cem\u003eIris\u003c/em\u003e plastome developed molecular markers can help us better identify and understand the evolutionary history of \u003cem\u003eIris\u003c/em\u003e species in the future.\u003c/p\u003e","manuscriptTitle":"Plastome Analysis and Phylogenetic Reconstruction of Iris Speci es in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 14:14:20","doi":"10.21203/rs.3.rs-7909828/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-19T12:35:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-18T14:18:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-12T08:59:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-08T13:07:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-06T04:19:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"149620812116441656624702060360462459840","date":"2025-12-04T04:51:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53682236863354824604751448608619210746","date":"2025-12-04T04:40:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"128177093037425544321862950569842280103","date":"2025-12-03T09:40:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-02T18:44:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329635352103161563466223031377005822342","date":"2025-12-02T16:50:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"208305017999389191849915310371164196176","date":"2025-12-02T16:03:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125362733724695895967908218581225035563","date":"2025-12-02T15:37:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313343599628986285853858069873531887123","date":"2025-12-02T15:17:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175361899004912221599542645676532667400","date":"2025-12-02T15:03:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T15:01:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-01T21:34:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-12T06:29:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-11T09:56:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-11-11T09:50:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5abbd282-28db-460e-bce6-ef055cc4446f","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T17:08:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-04 14:14:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7909828","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7909828","identity":"rs-7909828","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}