Introduction

Leaf, flower, and pollen micromorphology has been informative for resolving taxonomic problems in angiosperms across various taxonomic levels1,2,3,4,5,6,7. In particular, leaf epidermal stomata, orbicules, and pollen exine ornamentation characters have been shown to possess systematic values when examined using both light microscopy (LM) and scanning electron microscopy (SEM) and may further be utilized to test phylogenetic hypotheses8,9,10,11,12,13. However, despite an increasing number of the leaf, flower, pollen, and seed micromorphological studies in Iridaceae14,15,16,17,18,19,20,21, leaf margins, stomatal occurrence, and orbicular traits have not been considerably challenged for the taxonomic delineation of two closely related species, Iris ruthenica and I. uniflora. Therefore, understanding their micromorphology may shed light on their taxonomic relationships.

The chloroplast is an essential organelle for photosynthesis, starch and fatty acid biosynthesis, and carbon fixation22,23,24. The length of photosynthetic vascular plant plastomes ranges from 120 to 200 kb and possess a quadripartite structure, with one large single copy (LSC) region, one small single copy (SSC) region, and two inverted repeat (IR) regions. In general, angiosperm plastomes contain 110–130 genes, including approximately 80 protein-coding genes, 30 transfer RNA (tRNA) genes, and four ribosomal RNA (rRNA) genes22. These plastomes possess highly conserved structures and gene content and exhibit low variation when compared to nuclear and mitochondrial genomes. However, variable plastome size, gene content, IR expansion or contraction, and structural arrangement have been reported25,26. Plastome sequencing can be useful for species classification and identification and high-resolution phylogenetic analysis27,28. Facilitated by next-generation sequencing (NGS), plastome data are increasingly utilized for the investigation of phylogenetic relationships and for the development of DNA barcode markers for low-taxonomic-level identification and the discrimination of controversial taxa29,30,31. However, even though complete plastome data are available for a variety of Korean irises32,33, neither the detailed comparative analysis of Iris plastomes nor the combination of such analysis with micromorphological analysis has been conducted in the genus Iris as it has been in other taxonomic groups34,35,36.

The genus Iris L. contains approximately 300 perennial species, which are distributed in temperate regions across the Northern Hemisphere, as well as a large number of infraspecific taxa15,37,38,39. Based on recent molecular phylogenetic analyses, the genus has been divided into six subgenera40,41,42,43,44,45, including Iris L. subg. Limniris (Tausch) Spach ser. Ruthenicae Diels, which contains two species, I. ruthenica Ker Gawl. and I. uniflora Pall. ex Link37,45,46. Interestingly, both I. ruthenica and I. uniflora are rare or endangered in Korea (Fig. 1), owing to their similar pharmacological effect as the herbal medicines or because of their popularity as ornamentals47,48. However, even though the infrageneric classification of Iris based on chloroplast DNA sequence data remains somewhat controversial33,38,45, the monophyly of ser. Ruthenicae is strongly supported by molecular evidence (whole plastome sequences33), chromosome number46, and external morphology37, and the series can be easily distinguished from other series in the genus37. Iris ruthenica is arguably most closely related to I. uniflora due to their similar morphology37,47. Regarding the cytological features, the two species have consistent chromosome numbers (2n = 42), and similar genome sizes (2.42 pg/1C in I. ruthenica; 2.46 pg/1C in I. uniflora)46. However, despite of the economic significance of the two species, genomic resources for the ser. Ruthenicae are still limited. Interestingly, some taxonomists considered I. uniflora as a synonym of I. ruthenica due to their high morphological similarity37,49, and such an example can be found in other taxonomic groups in the genus Iris50. Thus, comparison of the genomic data and micromorphology of the two taxa is needed as indicated in recent studies41,44,46,51,52.

Figure 1
figure 1

Morphology and habit of Iris species from Korea. (a) I. ruthenica; (b) I. uniflora. Insets in (a) and (b) show the enlarged image of the flowers of the investigated species. All photographs were obtained by Tae-Soo Jang.

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Accordingly, the aims of the present study were to (1) compare the macro/microscopic features of I. ruthenica and I. uniflora, (2) characterize and compare the complete de novo-assembled plastomes for both species, and (3) investigate the evolutionary relationships within Iris through phylogenetic analysis33.

Results

Leaf, flower, pollen, and orbicule morphological characters

The two species possessed similar floral traits (e.g., tepal color, shape, and size; Fig. 1 and Supplementary Fig. S1) and similar leaf epidermis, epidermal cell, and anticlinal wall shapes (Fig. 2). The leaf epidermis of both species consisted of long tabular cells, typically with elongated pavement cells (Fig. 2c,i), with undulate anticlinal walls (Fig. 2e,k) that were covered by either prominent (I. ruthenica; Fig. 2f) or weak (I. uniflora; Fig. 2l) epicuticular wax. However, the leaf margin of I. uniflora was clearly entire, whereas that of I. ruthenica was spiny with sharp stiff points (Fig. 2), and the leaves of I. ruthenica were hypostomatic (stomata are absent or extremely rare on the adaxial leaf surface while they are present on the abaxial leaf surface; Fig. 2c,d), whereas those of I. uniflora were amphistomatic (stomata are present on both adaxial and abaxial leaf epidermis; Fig. 2i–j). In addition, the width of guard cells ranged from 24.63–28.19 μm on the abaxial surfaces of I. ruthenica leaves and from 26.53–28.14 μm and 27.02–27.77 μm on the adaxial and abaxial surfaces of I. uniflora leaves, respectively (Tables 1, 2). SEM analysis revealed that stomata of both species were clearly sunken (Fig. 2f,l), and that they were all anomocytic lacking subsidiary cells, surrounded by four epidermal cells (Fig. 2d,i,j). Tepal epidermal cells had unicellular covered by a striated cuticle and slightly sunk stomata (Supplementary Fig. S1). Furthermore, the outer anther epidermal cells were polygonal in shape and covered by a striated cuticle in both species, I. ruthenica and I. uniflora (Fig. 3g,h,q,r). Both species possessed anthers with anomocytic stomata in the middle section, despite differences in stomatal density (Fig. 3h,r). The guard cell surfaces of I. uniflora were weakly wrinkled (Fig. 3i), whereas those of I. uniflora were smooth (Fig. 3s).

Figure 2
figure 2

Stereo, light, and scanning micrographs of leaves of Iris ruthenica (a–f) and I. uniflora (g–l). Serrate (a,b) and entire (g,h) leaf margin. Hypostomatic (stomata is absent on adaxial surface but present on the abaxial surface of leaf) (c,d) and amphistomatic (stomata present on both adaxial and abaxial leaf epidermis) leaves (i,j) with anomocytic stomata. (e,f, k,l) Detailed leaf epidermal cell shape and sunken stomata on the leaf surface.

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Table 1 Plant material information for micromorphological and molecular analyses.
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Table 2 Overview of leaf and pollen morphological characters of Iris ruthenica and I. uniflora examined using light microscope.
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Figure 3
figure 3

Stamen, pollen, and orbicule micromorphology of Iris ruthenica (a–i) and I. uniflora (j–s). (a–d, j–n) Stained fertile (a,b) or sterile (j–l) pollen grains and their detailed sexine ornamentation; (e,f, o,p) dissected anther (inner part; e, o) and the occurrence of orbicules (f, p), respectively; (g–i, q–s) outer epidermis of anthers (g, q) and the occurrence of stomatal complex (h,i,r,s), respectively. All photographs were obtained by Bokyung Choi.

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The pollen grains of both species were monads of 47.54–56.31 μm in polar length and 39.18–53.55 μm in equatorial diameter (Table 2). While both species yielded fertile pollen grains (Fig. 3a,k), sterile pollen grains were only found extremely rarely or absent in I. ruthenica (Fig. 3a–c), and, in Iris uniflora, sterile pollen grains were frequently encountered (Fig. 3j–l), which differed from the fertile pollen grains of both species in regard to equatorial width (Table 2), shape (subprolate to prolate, P/E = 1.19–1.49 vs. oblate-spheroidal to prolate-spheroidal, P/E = 0.94–1.12; Table 2), and exine ornamentation (irregularly microreticulate exine ornamentation vs. monosulcate with microreticulate ornamentation; Fig. 3c–d,l–n). The orbicules of both species were entirely fused with the inner locule anther wall, particularly at the tapetal membrane (Fig. 3f, p), and possessed almost identical morphology (i.e., density, size, shape, and surface details).

Plastome sequencing

Illumina MiSeq yielded 5.2 and 6.0 Gb raw paired-end (2 × 300 bp) reads for I. ruthenica and I. uniflora, respectively, and 2.8 and 4.5 Gb trimmed reads, thereby providing coverage of approximately 739.3 × and 1064.6 × , respectively (Supplementary Tables S6 and S7).

Plastome characteristics

Both plastomes exhibited the quadripartite structure typical of angiosperm taxa (Fig. 4), with total lengths of 152,275 and 152,282 bp, LSC region lengths of 82,301 and 82,307 bp, and SSC region lengths of 18,134 and 18,135 bp in I. ruthenica and I. uniflora, respectively, as well as an IR region of 25,920 bp in length in both species (Table 3, Supplementary Figs. S2–4). The plastome junction regions were validated through the generation of high-quality plastome sequences. The overall GC content of the two plastomes was 38.1%, with greater GC content in the IR regions of I. ruthenica and I. uniflora (43.3 and 43.2%, respectively) than in the LSC regions (36.3 and 36.3%) and SSC regions (32.2 and 32.1%). Both plastomes contained 115 genes (80 protein-coding, 4 rRNA, and 31 tRNA genes; Table 3), 18 intron-containing genes (16 with one intron and two with three introns), and duplicate genes (ndhB, trnI-GAU, and trnA-UGC) in the IR regions (Supplementary Table S9). Analysis of codon usage and anticodon recognition patterns indicated that the plastomes of I. ruthenica and I. uniflora contained 26,634 and 26,641 codons, respectively, and that leucine, isoleucine, and serine were the most abundant (Supplementary Fig. S2). Relative synonymous codon usage (RSCU) analysis indicated synonymous codon bias, with a high proportion of A or T in the third position. Most RSCU values indicated a similar pattern. RSCU values for arginine were usually high.

Figure 4
figure 4

Circular gene maps of Iris ruthenica and I. uniflora plastomes. Genes drawn inside the circle are transcribed clockwise, and those outside the circle are transcribed counterclockwise. The darker gray in the inner circle represents GC content.

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Table 3 Summary of the major characteristics of the chloroplast genomes of I. ruthenica and I. uniflora.
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Plastome comparison

Plastome alignment revealed slight genomic variation, with intergenic regions being the most divergent. However, the plastomes generally formed a well-conserved collinear block (Supplementary Fig. S3), with a highly conserved structure. Nucleotide diversity analysis identified 14 regions with weak variation (Figs. 5, 6). The genes psbA and ycf1 yielded Pi values of 0.00188 and 0.00112, respectively. Most of the divergent regions were located in the LSC region (Pi = 0.00243). In particular, the trnK-matK, matK-trnK, and trnK-rps16 regions have shown consecutive nucleotide variations in the LSC region. The analysis of IR boundaries revealed that rpl22 was located in the LSC region of both species. The ycf1 and ndhA genes were located in the junction of SSC/IRb region. The rps19 gene, which was duplicated in the IR regions, was generally well-conserved. The ycf1 gene was located at the IRa/SSC and SSC/IRb junctions (Supplementary Fig. S4).

Figure 5
figure 5

Comparison of Iris ruthenica and I. uniflora plastomes using mVISTA. Complete plastomes of I. ruthenica and I. uniflora were compared to that of I. ruthenica. Blue block: conserved genes; sky-blue block: transfer RNA (tRNA) and ribosomal RNA (rRNA) genes; red block: conserved non-coding sequences (CNS). Regions with sequence variation between I. ruthenica and I. uniflora are denoted in white. Horizontal axis indicates coordinates within plastomes. Vertical scale represents percent identity, ranging from 50–100%.

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Figure 6
figure 6

Comparison of nucleotide diversity (Pi) values among Iris ruthenica and I. uniflora species and parsimony haplotype analyses using psbA, matK-trnK, and psbK-psbl.

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Phylogenic relationships among Iris species

The ML and BI topologies were highly congruent for the whole plastome and CDS datasets, and all but one lineage was strongly supported (ML > 95%, BI = 1.0). More specifically, the topologies were clearly divided into three major clades, which corresponded to the subgenera Limniris, Pardanthopsis, and Iris (Fig. 7). Most of the Iris species included in the present study were assigned to section Limniris, which is consistent with the Angiosperm Phylogeny Group (APG) IV classification system, except for I. tectorum53. Furthermore, a clade containing I. domestica and I. gatesii was clustered as a sister group to I. tectorum; I. ruthenica and I. uniflora formed a monophyletic clade (Fig. 7).

Figure 7
figure 7

Phylogenetic tree of Iris species constructed using maximum likelihood bootstrap analysis and Bayesian posterior probability. Maximum likelihood topology is shown with bootstrap support values and Bayesian posterior probabilities given at each node.

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Discussion

Micromorphology

Iris ruthenica and I. uniflora are sister taxa within ser. Ruthenicae and, as such, share a variety of morphological characters, including creeping rhizomes, leaf shape and size, flower color, and fruit shape37. Indeed, due to their morphological similarity, the taxonomic status of the species has been controversial. For example, Zhao et al.37 suggested that I. uniflora be considered a subgroup of I. ruthenica, and Zheng et al.54 argued that morphological differences between the species are the result of environmental conditions (i.e., different habitats). However, in the present study, I. ruthenica and I. uniflora could be differentiated using a variety of micromorphological features, including stomatal complex distribution (hypostomatic leaves vs. amphistomatic leaves), slight differences in the protruded conical cells of leaf surfaces, pollen grain fertility (all fertile vs. both fertile and sterile), and anther stomata density.

The micromorphological analysis performed in the present study revealed that I. ruthenica and I. uniflora possess leaves with an irregular shape and sinuate anticlinal striation, as previously reported14,20,55. Because both leaf surface micromorphology and stomata have shown considerable variation in papillae, they are not taxonomically significant, as suggested by Wu and Cutler14 and other monocot species56,57. The papillae that cover stomata likely contribute to defense against unfavorable environmental conditions58 or attack by pathogens59. Papillae can also play an important role in light reflection, by preventing overheating60, and, as such, may be more correlated with environmental conditions than taxonomic status. Regardless of stomatal distribution, both species possessed anomocytic stomata (Table 2), which is likely the ancestral (plesiomorphic) condition among monocots61. The guard cell and genome sizes of the two Iris species included in the present study were not significantly distinct (Table 2; Choi et al.46), although it is well known that stomata size is positively correlated with genome size and that ploidy level changes in plants62,63,64.

With the exception of clear differences in pollen viability, the morphology (e.g., shape, size, exine pattern, and orbicule presence) of pollen in I. ruthenica and I. uniflora were not significantly different (Fig. 3). As I. uniflora was only found under low-temperature stress conditions, whereas I. ruthenica was widely distributed46,47,48, the occurrence of sterile pollen grains in I. uniflora might be affected by meiotic abnormalities with genetic constitution as reported in other cold regions65,66. Orbicules, also known as Ubisch bodies, are small sporopollenin particles that can be produced on the inner locule walls of anthers67 and that are, here, reported in I. ruthenica and I. uniflora for the first time. Even though orbicule morphology has been widely used to elucidate systematic and evolutionary relationships12,68,69, the occurrence of orbicules has, until now, only been reported in I. pallida Lam.70. Thus, further investigation of orbicule occurrence in the Iridaceae might yield a significant phylogenetic trait, as reported in other plant groups67,68,69,70,71,72. However, as with other morphological features of Iris taxa, such as tepal structure17, pollen morphology15,16,21,73,74,75, and seed microstructure18, the micromorphological features of either vegetative or reproductive organs are largely insufficient for reconstructing the taxonomic relationship of I. ruthenica and I. uniflora.

Plastome characterization and variation

The structures of the I. ruthenica and I. uniflora plastomes were similar to those of plastomes from other members of the Iridaceae32,33, with the typical quadripartite structure and LSC and SSC regions separated by IR regions. The plastomes of the Iris species contained 114 unique genes, and their gene order, GC content, genomic structure, and overall length (152,275 and 152,282 bp) were within the ranges previously described for Iris plastomes76.

The mVISTA results indicated that the Iris plastomes contained little variation and that genic regions were more conserved than IGS regions, which is consistent with angiosperm plastomes in general77,78,79. More specifically, the psbA, trnK-matK, matK-trnK, and trnK-rps16 regions were hotspots for genetic variation (Fig. 5), which indicated underlying evolution80,81,82,83 and value as molecular markers84,85. In terms of nucleotide diversity (Pi), most of the divergent regions were non-coding, which is consistent with previous reports86,87,88. Other plastomes were highly variable for the non-coding regions at psbA, trnK-matK, matK–trnK, and trnK-rps16 in the present study (Fig. 6). mVISTA and Pi analysis showed difference between I. ruthenica and I. uniflora at the plastome level. Furthermore, these regions will play an important role in the discrimination of I. ruthenica and I. uniflora, as well as other species within the Iridaceae.

IR contraction and expansion causes variation in the size of angiosperm plastomes89. Previous studies have reported extremely short IRs or the loss of IR regions and genes90,91. Compared to I. ruthenica, I. uniflora had a highly conserved IR length and gene positions. However, the rps19 genes of I. ruthenica and I. uniflora were located in the IRa region, and ycf1 was located in the IRa/SSC, region which overlapped with ndhF. Thus, the Iris plastome possessed an extended IR, as reported previously32,33.

Phylogenetic relationships in the Iridaceae

Plastid genome (i.e., plastome) sequences are valuable genomic resources for estimating phylogenetic relationships, particularly among closely related species and unresolved taxa4,27,31. The systematics of Korean irises have been widely discussed, and several molecular studies based on single molecular markers (e.g., psbA-trnH, trnL-F) and plastome structure have been performed in the past33,92. The topologies of phylogenetic trees based on whole plastome sequences in the present study are similar to those reported previously (Fig. 7, Supplementary Figs. S5–S7)33,41,45,52,92. In agreement with earlier results, I. ruthenica and I. uniflora were nested deeply within section Limniris ser. Ruthenicae. However, in the present study, the monophyly of section Limniris was compromised by the inclusion of I. tectorum (ser. Lophris), which was deeply nested with subgenera Pardanthopsis and Iris in both the ML and BI trees (Fig. 7). Due to insufficient taxonomic sampling, the results of the present study are not suitable for the discussion of inter-subgeneric relationships within Iris s.l. 44. Nevertheless, the present study provides important genetic resources for further studies within the genus, as well as micromorphological comparisons between two closely related species in a taxonomic context.

The results of the present study confirm that Iris section Limniris ser. Ruthenicae (formerly section Ioniris sensu Zhao et al.37) contains the two species, I. ruthenica and I. uniflora. Morphologically, I. uniflora is distinguished from I. ruthenica because of the presence of narrow leaves54, but the flowers of the species are difficult to distinguish. In Korea, I. ruthenica is widespread, while I. uniflora is restricted to alpine areas46,47,48. Based on the comparison of morphological and chloroplast genome data of the two species, it is doubtful whether I. ruthenica and I. uniflora can be recognized as independent species-level taxa, as suggested by Zheng et al.54. Thus, further studies on molecular and morphological analyses of the two species at population level are required to clarify the taxonomic status of the two taxa.

Conclusions

The present study provides detailed insights into the leaf and flower micromorphologies and plastome structures of the two closely related species I. ruthenica and I. uniflora. Micromorphological features, including leaf margins, stomatal complex distribution (hypostomatic vs. amphistomatic leaves), anther stomata density, and floral epidermis cell patterns, are somehow useful for distinguishing the taxa, despite that the drastic influences of environmental variation, especially climate factors (e.g., temperature and light intensity), may also contribute to the morphological variations. The plastome sequences of the two related species possessed similar genome lengths, gene numbers, and gene orientations. Most of the variable sequences, which were found in non-coding regions, could be used as molecular markers for the differentiation of I. ruthenica and I. uniflora, as well as other Iris taxa. Given the economic and ecological importance of Korean Iris species, the molecular phylogenetic studies can now prompt a search for diagnostic characters, such as general morphological and micromorphological traits, a necessary prerequisite for any systematic and taxonomic context. Further integrative analyses of plastome sequences and morphological data of the two species at population level as well as employing molecular cytogenetic approaches using species-specific satellite DNA as probes may also offer insights into the species delimitation of the two closely related Iris species.

Materials and methods

Taxon sampling

All plant materials were collected from natural populations in Korea (Table 1). Assoc.-Prof. Dr. Tae-Soo Jang and Dr. Soonku So formally identified all the samples. Considering the protection of Korean endangered plant resources, we only collected a small number of plant specimens with the approval and permission of the local authorities (collection permit nos. 2019–13 [JC532], 2019–14 [BKC939], 2019–20 [JCKC190507]; Table 1). To evaluate the consistency of morphological and micromorphological characters, living specimens were collected from multiple populations (three I. ruthenica specimens from three populations and three I. uniflora specimens from one population), and cultivated at Chungnam National University (Table 1). Meanwhile, for plastome sequencing, representative fresh leaves were collected from I. ruthenica and I. uniflora specimens (accession numbers: BKC939 and JCK2019-77, respectively; Table 1). Specific locality information including GPS coordinates, latitude, and longitude cannot be provided due to the endangered/rare status of the species in Korea. All voucher specimens were deposited in the Chungnam National University Herbarium (CNUK).

Micromorphological analysis

Fresh leaf and flower materials from all four Iris populations were preserved using a formalin-acetic acid-alcohol solution and dehydrated by soaking in an acetone series (50, 70, and 90%) for 30 min and absolute acetone for 1 h. The dehydrated materials were then immersed in carbon dioxide for critical point drying (EMCPD300, Leica Microsystems, Germany), coated using an ion-sputtering device (E-1010, Hitachi, Japan), and analyzed using scanning electron microscopy (SEM; S3000N, Hitachi, Japan), with an accelerating voltage of 20 kV and working distance of 9–15 mm, following Choi et al.5, as well as light microscopy (BX53F, Olympus, Japan), which was used to analyze the structure of leaf epidermal pavement cells and stomatal complexes, following Kim et al.7. At least 20 guard cells were examined on both adaxial and abaxial leaf surfaces from each sample as described by Choi et al.5. To measure the pollen viability of each species, 10 randomly selected anthers from each individual plant were placed in an aniline blue dye solution to distinguish fertile pollen grains as described by Jang et al.93. At least 20 sterile and fertile pollen grains from each sample were randomly selected for size measurements. Both leaf and pollen micromorphological characters were measured using MicroMeasure ver. 3.3 program following Jang et al.94. For SEM, imaging was performed for both the abaxial and adaxial leaf surfaces, as well as for both the inner and outer surfaces of the outer tepals, stamens, and pollen grains. Orbicules, which are cellular structures of sporopollenin particles produced by the secretory tapetum, were investigated in this study for the first time using SEM in the genus Iris.

Plastome sequencing and assembly

Total genomic DNA was extracted from freshly collected samples using the modified CTAB method95, and Illumina short-insert paired-end sequencing libraries (TruSeq DNA Nano kit, Illumina, San Diego, CA, USA) were constructed and sequenced using the Illumina MiSeq platform. For de novo plastome assembly, sequencing reads were trimmed and filtered using FastQC v0.11.796, and the resulting trimmed paired-end reads (Phred score ≥ 20) were assembled using Velvet 1.2.1097, with kmer values of 71, 91 101, and 111 to form large contigs. The Velvet contigs were then assembled into complete plastomes using the de novo assembly option in Geneious prime (https://www.geneious.com) and ordered using reference plastome sequences from I. gatesii (NC_024936), I. sanguinea (NC_029227), and I. missouriensis (NC_042827). Finally, the LSC/IR, IR/SSC, SSC/IR, and IR/LSC regions of the complete plastomes were validated using PCR-based sequencing. Primer information and sequence alignment results are listed in Supplementary Tables S1 and S2.

Plastome annotation and repeat sequence analysis

The I. ruthenica and I. uniflora plastomes were annotated using GeSeq98. Protein-coding sequences were manually curated and confirmed using Artemis99 and then checked against the NCBI protein database. tRNA genes were confirmed using tRNAscan-SE 1.21100, and IR region sequences were confirmed using IR finder and RepEx101. Finally, circular maps of the I. ruthenica and I. uniflora plastomes were generated using OGDRAW102.

For plastome comparison, GC content and relative synonymous codon usage (RSCU) were calculated using MEGA6103. The two plastomes were also compared using mVISTA in Shuffle-LAGAN mode, with I. ruthenica plastome as a reference, and nucleotide variation (Pi) among the plastomes, excluding regions of < 200 bp, was calculated using DnaSP version 6.1104. Each plastome was divided into genes, introns, and intergenic regions.

Plastome phylogenetic analysis

The plastome sequences of eight taxa, including six other taxa from the Iridaceae and the outgroup taxa Crocus cartwrightianus (NC_041459) and C. sativus (NC_041459), were obtained from NCBI GenBank (Supplementary Table S3), and two matrices, one of whole plastome sequences and another of 78 conserved protein-coding sequences (CDS), which excluded duplicate genes in the IR region, were generated using MAFFT ver. 7105; all ten plastome sequences were then manually adjusted using Bioedit106. For CDS analysis, the aligned CDS were extracted and concatenated using Geneious (https://www.geneious.com) and filtered to remove ambiguously aligned regions using GBLOCKS ver. 0.91b107. The best-fitting model for nucleotide substitution was determined using the Akaike Information Criterion (AIC) in JModeltest V2.1.10108 (Supplementary Table S4). Maximum likelihood (ML) analysis was performed using RaxML v8.0.5109, with 1000 bootstrap replicates and the GTR + I + G model, and Bayesian Inference (BI) analysis was performed using MrBayes 3.2.2110, with two independent runs and four simultaneous Markov Chain Monte Carlo runs of 5,000,000 generations each. The resulting trees were sampled every 100,000 generations, with the first 25% discarded as burn-in, and the 50% majority-rule consensus tree was visualized using Figtree V.1.4.2111, with posterior probability (PP) values estimated from trees sampled after the burn-in fraction was discarded.

Ethics approval

The experimental research and field studies on plants, including the collection of plant material, complied with relevant institutional, national, and international guidelines and legislation. The appropriate permissions and/or licenses for collection of plant were obtained for the study.