Phylogenetic study of Iris plants in China based on chloroplast matK gene and nuclear ITS gene

doi:10.21203/rs.3.rs-4992391/v1

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Phylogenetic study of Iris plants in China based on chloroplast matK gene and nuclear ITS gene

https://doi.org/10.21203/rs.3.rs-4992391/v1

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Using both Neighbor-Joining and Maximum Likelihood algorithms, the ITS and matK sequences of Iris species in China were sequenced to explore the phylogenetic relationships of the six subgenera of Iris in China. After alignment of the ITS and matK sequences, a total of 893 characters were obtained for the ITS sequence, with a length variation range of 704–824 bp and 890 characters for the matK sequence, with a length variation range of 865–887 bp. The clustering analysis results were generally consistent with the subgenus classification of Iris species in China. Results suggested Subgen. Iris has a relatively complex phylogenetic relationship within the genus, and further research is needed to refine the internal classifications. Subgen. Limniris is resolved as polyphyletic and the phylogenetic relationships are quite complex. Subgen. Nepalensis is a relatively natural taxonomic group. Subgen. Crossiris is an unnatural taxonomic group. It is recommended to remove Subgen. Xyridion from the Iris classification system and to redefine the Belamcanda chinensis as a species within Subgen. Pardanthopsis of the Iris genus.

Iris L. matK ITS Phylogeny Taxonomic

Iris L. was established by Linnaeus in 1735 and is the largest and most evolved genus within the Iridaceae family. All species within the genus are perennial herbs, with the type species being Iris germanica L., which is native to Europe (Li et al. 2020). This genus exhibits high heterogeneity, with species displaying a wide variety of plant sizes, flower shapes and colors, which have high utilization value (Bar-Lev et al. 2021). Iris species have become significant models for phylogenetic studies and introgressive hybridization and have also been used in garden construction, ecological restoration and medicine (Arnold et al. 1990; Mocan et al. 2018; Kostić et al. 2019). In the past, Waddik and Zhao compiled and classified the distribution of irises in China. Iris genus has abundant germplasm resources, with about 300 species worldwide, around 60 species and 13 varieties and 5 forms in China. The southwestern region has the highest distribution, followed by the northwest and northeast, with fewer occurrences in the south (Waddick and Zhao 1992).

The classification of Iris plants began in the early 19th century with the system developed by Roamer and Schultes, the classification was mainly based on the observation of whether there were beards on the outer perianth of Iris and the Iris plants were divided into three groups according to this characteristic (Zhong 2010). Subsequently, many scholars have used the root characteristics, the presence of beard-like structures and crest-like decorations or combined the external features of fruits and seeds to define and classify the taxonomy of Iris plants (Dykes 1913; Lawrence 1953; Rodionenko 1987). Because of differences in delimitation and emphasis, there are a number of different classification systems for Iris plants under different criteria. Building on the integration and revision of previous research, Mathew (1989) resummarized the classification system of Iris genus, including bulbous Iris into the Iris genus. The genus is divided into six subgenera, twelve sections and sixteen series. Mathew's classification system is current relatively recognized internationally. Zhao’s (1985) classification of the Iris genus in China primarily references Rodionenko’s classification system, maintaining Rodionenko’s division of Chinese Iris species into six subgenera. Zhao made some adjustments and additions to some species based on particular circumstances and removed out series from sections under the subgenera. Zhao added sect. Ophioiris into the Subgen. Limniris. This system has been further revised in subsequent studies (Zhao et al. 2000) ( Table 1). With the improvement of research methods and continuous advancements in technology, the classification system of Iris species is no longer simply divided and defined based on external morphological characteristics. It has progressively evolved towards cytology, palynology and molecular biology, with researchers now beginning to conduct comprehensive system analyses combining multiple fields (Ikinci et al. 2011; Azimi et al. 2012; Mitić et al. 2013; Wilson et al. 2023).

Table 1

Subgenera and sectional divisions in different *iris* classification systems
Classification of Iris L. of Rodionenko(1987)	Classification of Iris L. of Mathew(1989)	Classification of Iris L. of Y. T. Zhao(1985)
Subgen. Limniris	Subgen. Limniris	Subgen. Limniris
Sect. Ioniris	Sect. Limniris	Sect. Ioniris
Sect. Limniris	Sect. Lophiris	Sect. Limniris
Sect. Unguiculares	Subgen. Iris	Sect. Ophioris
Subgen. Iris	Sect. Iris	Subgen. Crossiris
Sect. Iris	Sect. Oncocyclus	Sect. Crossiris
Sect. Hexapongon	Sect. Psammiris	Sect. Lophiris
Subgen. Crossiris	Sect. Reglelia	Subgen. Iris
Sect. Crossiris	Sect. Hexagonae	Sect. Iris
Sect. Lophiris	Sect. Pseudoregelia	Sect. Hexapogon
Sect. Monospatha	Subgen. Xiphium	Subgen. Xyridion
Subgen. Pardanthopsis	Subgen. Nepalensis	Subgen. Nepalensis
Subgen. Nepalensis	Subgen. Juno	Subgen. Pardanthopsis
Sect. Ophioiris	Subgen. Iridodictyum
Sect. Xyridion	Sect. Brevituba
Sect. Spathula	Sect. Monospatha
	Sect. Hermodactyloides
	Sect. Micropongon

Molecular biology utilizes the molecular data of biological genetic information, with the help of statistical methods for quantitative descriptive analysis. This approach is used to study issues of genetic diversity, growth and development patterns and system evolution at the molecular level (Reeves et al. 2001; Goldblatt et al. 2008). DNA sequencing includes sequencing of ribosomal DNA (rDNA), chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA). Among these, rDNA evolves the fastest, followed by cpDNA with mtDNA evolving the slowest. Within the genome, the evolution rate of coding regions is lower than that of non-coding regions (Martínez et al. 2010; Park et al. 2022). The nuclear genome, inherited from both parents, contains a vast amount of genetic information and its sequence variations are conducive to resolving the issues of reticulate evolution (Packia et al. 2024). The internal transcribed spacer (ITS) regions include the ITS1 region, the 5.8S region and the ITS2 region. This gene is subject to minimal natural selection pressure during the evolution of higher plants but evolves rapidly. The nucleic acid sequences among different species exhibit high variability and rich polymorphism, providing abundant variant sites and informative loci for closely related species (Rajesh et al. 2024). It plays a significant role in analyzing genetic diversity, studying phylogeny and identifying species (Baldwin et al. 1995; Wilson 2003). The matK gene is located within the intron of the trnK gene, is approximately 1500 bp and encodes a maturase (Sevindik et al. 2024). It is one of the fastest-evolving genes in the protein-coding region of the chloroplast genome. This gene can provide substantial information for studying intra-population phylogenetic relationships and has a high support rate, making it highly important for exploring inter-genus and intra-genus relationships in molecular biology (Neuhaus and Link 1987).

Although extensive systematic research has been conducted on Iris genus in terms of morphology, cytology, palynology and molecular biology, there is still no consensus on the systematic classification and some controversial species (Samad et al. 2016; Choi and Lee 2024). In this study, we collected, constructed and analyzed gene trees representing six subgenera of Iris and three outgroup species to explore the interspecific relationship among Iris species, redefine the classification status of controversial species and discuss the systematic classification of Iris plants.

Plant materials

For this study, thirty samples of 3 outgroup taxa and 27 species representing five subgenera of the Iris L. were collected from Sichuan, Gansu, Yunnan and Chongqing in China (Table 2). All the plants were identified by Professor Yonghong Zhou and Associate Professor Xiaofang Yu from Sichuan Agricultural University, with the voucher specimens stored in the herbarium of the Wheat Research Institute at Sichuan Agricultural University.

Table 2

*Iris* materials collected in the wild for experiments
Formal name	Altitude(m)	Locality
Iris wilsonii C. H. Wright Iris forrestii Dykes Iris sibirica L. Iris bulleyana Dykes Iris delavayi Mich. Iris pseudacorus L. Iris lactea Pall. Iris lactea var. chinensis(Fisch)Koidz Iris lactea var. chrysantha Y. T. Zhao Iris songarica Schrenk Iris ruthenica var. nana Maxim. Iris halophile Pall. Iris collettii Hook. f. Iris decora Wall. Iris speculatrix Hance. Iris japonica Thunb. Iris japonica f. pallescens Iris confusa Sealy Iris wattii Baker Iris tectorum Maxim. Iris germanica L. Iris pandurata Maxim. Iris sichuanensis Y. T. Zhao Iris leptophylla Lingelsheim Iris goninocarpa Baker Iris goninocarpa var. grossa Y. T. Zhao Iris goninocarpa var. tenella Y. T. Zhao Gladiolus gandavensis Van Houtte Crocosmia crocosmiflora N. E. Br. Belamcanda chinensis (L.) DC.	2452 2452 2011 3604 3203 1754 2160 855 3234 3363 2469 3316 1182 1177 1419 620 2001 1782 1973 2652 2011 2080 1358 1788 3034 2643 3213 1819 979 474	Mianning, Sichuan, China Mianning, Sichuan, China Kunming Institute of Botany, Yunnan, China Shangri La, Yunnan, China Dali, Yunnan, China Yanyuan, Sichuan, China Jinchuan, Sichuan, China Wenchuan, Sichuan, China Xiahe, Gansu, China Ruoergai, Sichuan, China Kangding, Sichuan, China Ruoergai, Sichuan, China Luding, Sichuan, China Luding, Sichuan, China Nanchuan, Chongqing, China Pujiang, Sichuan, China Dujiangyan, Sichuan, China Luding, Sichuan, China Dujiangyan, Sichuan, China Kangding, Sichuan, China Kunming Institute of Botany, Yunnan, China Wenchuan, Sichuan, China Wenchuan, Sichuan, China Wenchuan, Sichuan, China Ruoergai, Sichuan, China Maoxian, Sichuan, China Ruoergai, Sichuan, China Hanyuan, Sichuan, China Qianwei, Sichuan, China Chengdu, Sichuan, China

DNA amplification and sequencing

Leaf samples were collected from the wild or from living specimens in the Sichuan Agricultural University nursery and frozen at -80°C. Total DNA was extracted from 50–100 mg of stored leaf samples using a modified CTAB (Cetyltrimethylammonium bromide) method (Mishra et al. 2019). The genomic DNA precipitated with ethanol was dissolved in 30-50ul of TE solution or ddH2O and stored in a -20°C freezer for later use. The ITS sequences were amplified using universal primers F (5'-TCGCGAGAAGTCCACTGA − 3') and R (5'-ATGCTTAAACTCAGCGGG-3'). The ITS sequence-specific primers from researcher Wang (2005) included parts of the 18S region, the entire ITS1 region, the 5.8S region, the ITS2 region and parts of the 26S region. The universal primers for the matK sequences referring to Zhong’s (2010) method were F (5'-CCTATCCATCTGGAAATCTTAG-3') and R (5'-GTTCTAGCACAAGAAAGTCG-3'). DNA amplification was performed in a 50 µL reaction mixture with 50 ~ 100 ng of template DNA (Table 3). The PCR reaction for ITS was as follows: 1 cycle at 94°C for 3 minutes, 34 cycles of 30s at 94°C, 30s at 53°C, 1 minute 30s at 72°C, with a final extension at 72°C for 10 minutes. The PCR reaction for matK was as follows: 1 cycle at 94°C for 4 minutes, 34 cycles of 30s at 94°C, 30s at 52°C, 2 minutes at 72°C, with a final extension at 72°C for 10 minutes. The amplified double-stranded DNA fragments were detected by electrophoresis on a 1.5% agarose gel and visualized using the Gel Doc™ XR⁺ with the Image Lab™ software(Bio-Rad, Hercules, CA, USA). The products were purified using the DNA Gel Extraction Kit according to the manual and then cloned into the pMD19-T vector (TaKaRa) following the manufacturer's instructions. Six to ten positive clones of the ITS gene and three to five positive clones of the matK gene were selected from each material and sent to the Sangon Biotech (Shanghai, China) for bidirectional sequencing.

Table 3

Amplification system of ITS gene and *matK* gene
ITS amplification system		matK amplification system
Reaction system Dosage		Reaction system Dosage
Buffer Mg²⁺ dNTP DNA ex-Taq Primer F Primer R ddH₂O DMSO	5 µl 4 µl 4 µl 4 µl 1 µl 1 µl 1 µl 27.5 µl 2.5 µl	Buffer Mg²⁺ dNTP DNA ex-Taq Primer F Primer R ddH₂O	5 µl 4µl 4 µl 4 µl 1 µl 1 µl 1 µl 30 µl

Phylogenetic data analysis

The sequencing results were compared using the Blast software in NCBI and the sequences were proofread and assembled with DNAman 8.0. Multiple sequence alignment was then performed using MAFFT with appropriate manual corrections. All sequences intended for phylogenetic analysis underwent saturation testing and format conversion. Finally, phylogenetic analysis was conducted using MEGA6.0 software (Tamura et al. 2013), including Conserved sites (C), Parsimony information sites (Pi), Variable sites (V), Singleton sites (S), Nucleotide composition, Nucleotide transitions and transversions and their ratios (Si/Sv). For the 27 species of Iris, both Neighbor-Joining (NJ) and Maximum Likelihood (ML) algorithms were used to construct phylogenetic trees with the ITS and matK sequences. All bases in the arranged sequences were equally weighted and treated as unordered characters, with gaps treated as missing data. The analysis was performed using the Maximum Composite Likelihood method and the Bootstrap method, which cycles 1000 times. Topological robustness NJ analysis was assessed by bootstrap analysis with 1000 replicates (Tamura et al. 2013).

ITS and matK sequence analysis

Using the sequences uploaded by Wang (2005) in GenBank, we analyzed the ITS region segments of Iris species to determine the locations of different segments in the sequences. After aligning and sorting the ITS and matK sequences from 30 samples (including three outgroup taxa), we obtained a total of 893 characters for the ITS sequences, with the length variation ranging from 704 to 824 bp. The ITS1 + 5.8S + ITS2 segment had a length variation between 574 and 694 bp, showing significant length variation with insertions and deletions. The matK sequences had a total of 890 characters, with a length of all sequences varying from 865 to 887 bp and some regions also showed insertions and deletions. By analyzing the alignment matrix data, we found that the ITS total sequence, including outgroup taxa, had 343 conserved sites and 523 variable sites, with major variations occurring in the ITS1 and ITS2 regions. The matK total sequence, including outgroup taxa, had 661 conserved sites and 226 variable sites (Table 4).

The base substitution frequency in the ITS sequence and the matK sequence is higher than the base transversion frequency. The average G + C content in the ITS sequence is higher than the average A + T content, while the average G + C content in the matK sequence is lower than the average A + T content (Table 5). Saturation tests were performed on all ITS and matK sequences separately in DAMBE and the results did not reach saturation, indicating that they can be used to construct phylogenetic trees. The genetic distances calculated in Mega were all less than 1, suggesting that NJ trees can be constructed.

Table 4

Base composition of ITS and *matK* sequences of 30 species of Iridaceae
Gene	T (%)	C (%)	A (%)	G (%)	A + T (%)	G + C (%)
ITS	15.8	32.8	19.6	31.8	35.4	64.6
matK	37.8	16.6	30.0	15.5	67.8	32.1

Table 5

Characters of ITS and *matK* sequences of 30 species of *Iris* L.(including outgroup)
gene	Conserved	Variable	parsimony-infor	Singleton	Si	Sv	R = si/sv
ITS	343	523	334	184	47	41	1.1
matK	661	226	148	78	16	7	2.1
Note: si = Transitionsal Pairs、sv = Transversional Pairs

Phylogenetic analysis of Iris Species Based on ITS Sequences

The results obtained ITS sequences from 30 accessions, including outgroup species: Crocosmia crocosmiflora N. E. Br., Gladiolus gandavensis Van Houtte and Belamcanda chinensis DC.. Combined with ITS sequences of four Iris species from China downloaded from GenBank (Table 6), MEGA6.0 was used to construct Neighbor-Joining (NJ) (Fig. 1) and Maximum Likelihood (ML) (Fig. 2) phylogenetic trees and the phylogenetic tree was supported by the bootstrap test (³50%). The 31 Iris taxa represent five subgenera of Chinese Iris: the Subgen. Limniris, the Subgen. Iris, the Subgen. Crossiris, the Subgen. Xyridion and the Subgen. Nepalensis. The clustering results based on the ITS-constructed NJ (Fig. 1) tree and ML (Fig. 2) tree are generally the same, with minor differences observed in the positions of a few species, so the analysis will primarily use the NJ tree as an example.

The G. gandavensis and the C. crocosmiflora cluster together (Outgroup), forming a distinct branch on their own, indicating that they are more distantly related to the Iris genus. B. chinensis is embedded within the Iris genus (Clade B). The Iris genus is divided into five branches: Clade A1 and Clade A2 consist of species within the Subgen. Iris, with Clade A1 being positioned at the outermost part of the Iris species and this subgenus is also divided into several parts in the ML tree; the third branch (Clade B) is composed of the Subgen. Nepalensis. and B. chinensis; the fourth branch (Clade C) consists of species within the Subgen. Crossiris. The fifth branch (Clade D) includes all species belonging to the Subgen. Limniris. Surprisely, the I. halophila from the Subgen. Xyridion and the I. speculatrix from the Subgen. Crossiris are also embedded in it. The I. songarica is clustered on the outermost edge of the ML tree, but its support rate is very low. Considering the characteristics of its outer floral segments: lacking appendages, we agree with the clustering results of this species in the NJ tree. Among the 31 groups, except for the I. songarica and the I. speculatrix, the clustering results of other species generally align with Zhao’s (1985) classification system.

Table 6

List of accession and information of ITS sequences of *Iris* downloaded from GenBank
Species	Voucher	Locality	Accession No.
Iris ensata Thunb. Iris sanguinea Donn et Horn. Iris laevigata Fisch Iris mandshurica Maxim.	Liu Mingyuan Zhuo Lihuan, Wang Ling Zhuo Lihuan, Wang Ling Liu Mingyuan	Haerbing, China shenyang botanical garden, China Hailin, China Haerbing, Chia	DQ277637 DQ277636 DQ277643 DQ277642

Phylogenetic analysis of Iris species based on matK sequences

The result obtained the matK sequences from 30 materials, including outgroup species: C. crocosmiflora, G. gandavensis and B. chinensis and downloaded 42 matK sequences of Iris species (including varieties) from GenBank (Table 7). Using MEGA6.0, Neighbor-Joining (NJ) (Fig. 3) and Maximum Likelihood (ML) (Fig. 4) phylogenetic trees were constructed, removing all bootstrap test below 50%. 69 groups of Iris species represent the six subgenera of Chinese Irises: Subgen. Limniris, Subgen. Iris, Subgen. Crossiris, Subgen. Xyridion, Subgen. Nepalensis and Subgen. Pardanthopsis. The clustering of the NJ tree (Fig. 3) and the ML tree (Fig. 4) based on matK sequences is generally consistent, with only minor differences in the positions of a few species. The analysis in this study is primarily based on the NJ tree.

C. Crocosmiflora and G. gandavensis were clustered together as an outgroup, indicating their distant relationship with the genus Iris, while B. chinensis was nested within the genus Iris (Clade C). The genus Iris was divided into several clades. Species within the Subgen. Limniris were not clustered into a single large clade but were divided into three clades (Clade A1, A2, A3). In the ML tree, A2 and A3 were grouped together, dividing the subgenus into two clades. Additionally, I. halophila and I. halophila var. sogdiana of the Subgen. Xyridion (Clade A1) and I. proantha var. valida (Clade A2) and I. speculatrix (Clade B1) of the Subgen. Crossiris are also nested within the subgenus, indicating that the Subgen. Limniris is not monophyletic. The sepal of irises in Clade B2 are adorned with crest-like appendages, which include all species in the Subgen. Nepalensis and most species in the Subgen. Crossiris. Among them, I. subdichotoma from the Subgen. Pardanthopsis, as well as I. latistyla, I. tectorum and I. tectorum f. alba from the Subgen. Crossiris, were also integrated into the Subgen. Nepalensis. However, in the ML tree, I. tectorum and its variant still clustered within the Subgen. Crossiris. I. dichotoma from the Subgen. Pardanthopsis and B. chinensis cluster together (Clade C) and formed a separate branch. All collected irises belonging to the Subgen. Iris in this study clustered together (Clade D) but were divided into several subclades. Additionally, I. ventricosa from the Subgen. Limniris was also integrated into this subgenus. Among the 69 groups, except for I. halophila, I. halophila var. sogdiana, I. proantha var. valida, I. speculatrix, I. subdichotoma, I. latistyla, I. tectorum, I. tectorum f. alba and I. ventricosa, the clustering of the remaining species is generally consistent with Zhao’s (1985) classification system.

Table 7

List of accessions and information of *matK* sequences downloaded from GenBank
Species	Voucher	Locality	Accession No.
Iris typhifolia Kitagawa Iris ensata Thunb. Iris chrysographes Dykes Iris sanguinea Donn et Horn. Iris bulleyana Dykes Iris bulleyana f. abla Y. T. Zhao Iris clarkeri Baker Iris laevigata Fisch Iris minutoaurea Makino Iris rossii Baker Iris henryi Baker Iris setosa Pall. ex Link Iris tenuifolia Pallas Iris qinghainica Y. T. Zhao Iris loczyi Kanitz Iris songarica Schrenk ex Fische Iris ventricosa Pall. Iris ruthenica Ker-Gawl. Iris uniflora Pall. ex Link Iris uniflora var. caricina Iris anguifuga Y. T. Zhao Iris dichotoma Pall. Iris subdichotoma Y. T. Zhao Iris formosana Ohwi Iris latistyla Y. T. Zhao Iris tectorum f. alba Makino Iris milesii Baker ex M. Iris proantha var. valida(Chien)Y. T. Zhao Iris pallida var pallida Lam. Iris mandshurica Maxim. Iris potaninii Maximowicz Iris potaninii var. ionantha Y. T. Zhao Iris potaninii var. potaninii Iris tigridia Bunge ex Ledebour Iris bloudowii Ledebour Iris flavissima Pall. Iris kemaonensis D. Don Iris scariosa Willdenow ex Link Iris collettii var. acaulis Iris barbatula Noltie ex K.Y. Guan Iris odaesanensis Y.N. Lee Iris halophile var. Sogdiana Grubov	C. Wilson DBG05-35, RSA N. Alexeeva 02_EN_07, RSA Wilson PH04-12, RSA ZhouSL-kunming-Z083 Wilson PH04-10, RSA z213 Betty Ford Alpine Gardens 2005 − 111 UCBG 2001. 0082, UC D.K. Kim 08-124 ZhouSL-dandong-Z111 Guo JW11-35, RSA A. Wheeler ,WA_01_SE_07, RSA ZhouSL-qitai-Z088 - ZhouSL-bole-Z091 Missouri Botanic Garden 1989–7061 ZhouSL-heilongjiang-Z015 ZhouSL-xizang-Z223 Guo Bj07-23, RSA ZhouSL-shenyang-Z216 ZhouSL-eryuan-Z212 Wilson, DBG05-34, RSA z102 Chung 1928 HAST Guo BJBG07-32, RSA Guo BJBG07-28, RSA Guo CH10-30, RSA Guo HZ08-17, RSA Wilson LB05-46, RSA Wilson DBG05-19, RSA N. Alexeeva, R01-22, HPSU Wilson BJ07-27, RSA Wilson R01-22, RSA Wilson R01-18, RSA z092 Wilson Mg04-14, RSA Wilson NP06-65, RSA z090 Chase-I-131 Wilson PH04-11 Wilson JPW05-01, RSA Goldblatt & Porter 12233, MO	- - - China - Yunnan, China - - - China - - China Gansu, China China - China China - Yunnan, China China - Yunnan, China - - - - - - - Kyrgyzstan - - - Xinjiang, China - - Xinjiang, China China - - -	KC118942 KC118919 FJ197270 JF954173 FJ197269 KP089599 FJ197272 FJ197287 JF972932 JF954166 KC510987 KC118939 JF954183 KT280220 JF954158 FJ197299 JF954190 JF954168 FJ197309 JF954189 JF954117 HM574667 KP089633 KC510980 KC510982 KC510986 KC510983 HM574690 HM574689 HM574643 AY596654 HM574641 HM574642 HM574674 KP089598 HM574639 HM574646 KP089627 FR870426 HM574647 FJ197293 FJ197298

Taxonomic relationships of the subgenus Iris

In NJ tree based on ITS sequences (Fig. 1), the Subgen. Iris did not form a large branch but is divided into two branches (Clade A1, A2). Zhao (1985) classified this subgenus into two sections: Sect. Iris and Sect. Hexapogon. I. germanica in Sect. Iris clustered with I. pandurata and I. mandshurica in Sect. Hexapogon, while the others in Sect. Hexapogon grouped together. This is largely consistent with Wilson's (2017) research results, suggesting that there may be significant differences in the degree and timing of differentiation among species within the subgenus, indicating that the phylogenetic relationships within this subgenus need further study. I. goniocarpa var. tenella and I. goniocarpa formed a sister clade (99%), while I. goniocarpa var. grossa grouped with I. leptophylla, indicating that the relationship between I. goniocarpa and I. goniocarpa var. tenella was very close, whereas I. goniocarpa var. grossa was more distantly related to both. Observations reveal that the I. goniocarpa is only slightly more robust than I. goniocarpa var. tenella, with no significant differences in other aspects. I. goniocarpa var. grossa has a purplish-red flower color and the spots around the orange-yellow crest-like ornament on the outer tepals are larger and dark purple. The plant itself is larger and more erect, with broader and thicker leaves, distinctly different from the other two. Zhao et al. (2000) reclassified the I. goniocarpa var. grossa as I. cuniculiformis Noltie & K. Y. Guan, elevating it from a variety to a species. This study supports treating the I. goniocarpa var. grossa as a species and considers the I. goniocarpa var. tenella as a synonym of the I. goniocarpa.

In the gene tree constructed based on matK sequences (Fig. 3), all bearded irises clustered together (Clade D). I. pallida and I. germanica in the Sect. Iris formed a sister clade; both have rhizomatous roots and their seeds dehisce from the top of the fruit upon maturation. However, I. scariosa from the Sect. Hexapogon clustered with them, indicating a close phylogenetic relationship among the three. This is consistent with the findings of Zhong (2010). He speculated that this clustering result is due to their origin all in Central Asia and Europe and, they have identical chromosome number of x = 24. The remaining irises in the Sect. Hexapogon were divided into several clades, showing more complex phylogenetic relationships within the subgenus. Based on the results from both single-gene and multi-gene studies, Zhong (2010) also suggested that the subgenus classification is not uniformand. The phylogenetic relationships within the Subgen. Iris are complex and the division requires further study.

Taxonomic Relationships of the subgenus Crossiris

Subgen. Crossiris has 9 species and 2 varieties in China. In the ITS sequence phylogenetic tree (Fig. 1) clustering analysis, I. speculatrix was embedded in the Subgen. Limniris and formed a separate clade (Clade D), which is consistent with the results of molecular biology studies by Zhong (2010). Zhong suggested classifing the I. speculatrix into the Subgen. Limniris. Wu and Culter (2010) found that the leaf anatomical structure of I. speculatrix is similar to that of the Subgen. Limniris. Therefore, it is evident that its affinity with the Subgen. Limniris is relatively close. However, considering the characteristics of its sepal which has cristate ornaments, we do not support Zhong’s proposal to move this species into the Subgen. Limniris. Instead, we believe this species may be a transitional form between the Subgen. Limniris and the Subgen. Crossiris.

In the NJ tree based on the matK sequence (Fig. 3), except for I. proantha, for which no relevant sequence was collected, sequences for other species were collected. However, we collected the matK sequence for its variety I. proantha va,r. valida. Most species within the subgenus form a clade (Clade B2), but I. speculatrix, I. proantha var. valida, I. latistyla and I. tectorum are not clustered within this subgenus, indicating that this subgenus is not a natural taxon. I. speculatrix was embedded within the Subgen. Limniris and it forms a distinct clade (Clade B1) (73%), which is similar to the clustering result of this ITS analysis. I. latistyla is grouped into the subgen. Nepalensis (88%). This is consistent with the clustering results based on five chloroplast genes by Guo and Wilson (2013), who observed that the flower color and crest-like sepal ornament of this species are very similar to those of I. decora and I. collettii. Our study indicates that it is closely related to the subgen. Nepalensis, but its specific taxonomic position requires further data for exploration.

Taxonomic relationships of the subgenus Limniris

Zhao et al. (1985, 2000) divided species of the subgen. Limniris into three sections sect. Ioniris, sect. Limniris and sect. Ophioris. Our research also found that the subgen. Limniris exhibits multiple origins, as some elements of the sect. Limniris are located outside the core clade of the subgen. Limniris. In the ITS sequence NJ tree (Fig. 1), all species within this subgenus collected for this study cluster together (Clade D), but I. songarica is situated at the very edge of the subgenus, indicating it is more distantly related to the other species. I. ruthenica var. nana, I. lactea and its two varieties, as well as the I. halophila within the subgen. Xyridion, form a subclade. They are all relatively drought-tolerant or salt-alkali resistant. Zhao et al. (1985, 2000) classified I. lactea as the original species, with I. lactea var. chinensis and I. lactea var. chrysantha as varieties. However, Mathew (1989) argued that the floral color variations within this group are due to natural hybridization and that varieties should not be distinguished. Gao (1985) believed that I. lactea and I. lactea var. chrysantha are synonyms for I. lactea. In the NJ tree, this group clustered into a single clade(96%), indicating close relationships among the three. However, I. lactea and I. lactea var. chrysantha formed a sister clade(64%) and I. halophila was also embedded within this group, suggesting that the group is not a monophyletic origin. Our research results align with previous studies, supporting Zhao’s classification of I. lactea and I. lactea var. chrysantha as varieties of I. lactea as reasonable (Zhao et al. 2000).

Based on the matK sequence analysis, in the NJ tree (Fig. 3), the subgen. Limniris does not form a large clade but is instead divided into three clades (Clade A1, A2, A3), with species from other subgenera interspersed in between. This indicates that the subgenus is not a monophyletic group. The result was the same as that of in ITS-constructed NJ tree, I. halophila and its varieties are interspersed, forming a subclade with I. songarica and I. qinghainica and are clustered together with the sister clades of I. tenuifolia and I. loczyi (Clade A1). This is because these irises all grow in the relatively arid northwest region. I. odaesanensis, I. henryi, I. minutoaurea and I. rossii form a clade (Clade A2), generally consistent with Park's clustering based on the whole chloroplast genomes (Park et al. 2022). With the exception of I. henryi, all other species in this clade are native to the northeastern region. This clade also includes I. proantha var. valida from Subgen. Crossiris. Wilson (2009) believed that the phylogeny of the Subgen. Crossiris shows a certain geographic aggregation.

Taxonomic relationships of the subgenus Xyridion

In the phylogenetic tree based on ITS sequences(Fig. 1), I. halophila is the only species within the subgen. Xyridion. Mathew and Waddick suggested that it belongs to the subgen. Limniris (Mathew 1989; Waddick and Zhao 1992). In the NJ tree, this species is embedded within the subgen. Limniris (Clade D). This is consistent with the clustering results of multiple genes combined by Zhong (2010) and Mou (2011) also found that I. halophila grouped together with species within the subgen. Limniris through AFLP markers. The main characteristic of this subgenus is that the petals resemble a violin, but the midrib of the outer tepals lacks appendages, which aligns with the characteristics of the subgen. Limniris.

In the NJ tree of the matK sequence (Fig. 3), I. halophila var. sogdiana and I. halophila have been clustered into the subgen. Limniris, along with I. songarica and I. qinghainica, forming a subclade (Clade A1). This clade shares the same geographic distribution and similar external morphology. For example, both the I. halophila and I. songarica have violin-shaped petals, though the former has a sturdier flower stalk. The main difference between I. halophila and its variety lies in flower color: the former is yellow, while the latter can be blue-purple or variegated, with the claws of the inner and outer tepals being yellow and the upper parts being blue-purple. Based on the feature of having no appendages on the outer tepals, our findings are consistent with previous studies (Mathew 1989; Waddick and Zhao 1992; Zhong 2010): we recommend removing the species’ status as a separate subgenus and incorporating it into the Subgen. Limniris and we support Zhao’s treatment of I. halophila var. sogdiana as a variety of I. halophila (Zhao 1985).

Taxonomic Relationships of the subgenus Nepalensis

In the NJ tree constructed from the ITS sequence (Fig. 1), I. collettii and I. decora of the subgen. Nepalensis formed a sister clade (Clade B) (100%), indicating a close phylogenetic relationship. Both species exhibit the typical characteristics of the subgenus: fleshy spindle-shaped roots. We agree with previous research findings (Zhong 2010; Jiang et al. 2017) that this subgenus is a relatively natural taxonomic group. In the matK sequence study, the subgen. Nepalensis not only includes I. decora and I. collettii but also encompasses I. barbatula and I. collettii var. acaulis, which all cluster together (Clade B2) with 97% bootstrap support. The two newly added species also exhibit fleshy, spindle-shaped roots, consistent with the typical characteristics of this subgenus (Zhao et al. 2000).

Taxonomic relationships of the subgenus Pardanthopsis

The subgen. Pardanthopsis contains only two species, I. dichotoma and I. subdichotoma Y. T. Zhao. However, the classification status of I. dichotoma has always been controversial. Mathew (1989) believed that I. dichotoma should not be included in the Iris L. and classified it in the Pardanthopsis Lenz. Most scholars such as Baker, Dykes, Lawrence and Rodionenko (Baker 1892; Dykes 1913; Lawrence 1953; Rodionenko 1987) believed that I. dichotoma should be included in the Iris L. and placed it in the Subgen. Pardanthopsis. In the phylogenetic tree of the matK sequences, I. dichotoma was nested in the Iris L., indicating that I. dichotoma is closely related to the Iris L.. I. dichotoma together with Belamcanda chinensis, forms a separate clade, consistent with Wilson's clustering based on different genes and Feng's clustering based on complete chloroplast genomes (Wilson 2011; Feng et al. 2022). Comprehensive studies suggest that the classification status of the subgen. Pardanthopsis is reasonable. However, in the NJ tree (Fig. 3), I. subdichotoma is embedded in the subgen. Nepalensis, forming a sister clade with I. barbatula(66%). Zhong (2010) also found that this species clusters within the subgen. Nepalensis through multi-genome analysis. Based on palynological research, Qi and Zhao (1987) discovered that the pollen of I. subdichotoma is nearly spherical with a colpus of two-sulcus type, the exine stratification is not significant and it has a coarse reticulate ornamentation with granules within the meshes. These characteristics are similar to the pollen morphology of the subgen. Nepalensis and clearly distinguish it from the pollen morphology of the Subgen. Pardanthopsis: nearly spherical, single-sulcus colpus, distinctly two-layered exine, reticulate ornamentationand no granules within the meshes. Combining previous studies, we suggest transferring I. subdichotoma from the Subgen. Pardanthopsis to the Subgen. Nepalensis.

Classification of Belamcanda chinensis

In the NJ tree constructed from the ITS sequence (Fig. 1), Belamcanda chinensis was embedded within the genus Iris (Clade B) (70%). Baker, Dykes, Lawrence, Rodionenko and Mathew considered Belamcanda chinensis should be classified under the genus Belamcanda (Baker 1892; Dykes 1913; Lawrence 1953; Rodionenko 1987; Mathew 1989). However, Tillie, Wilson, Goldblatt and Mabberley argued that it should be classified under the genus Iris based on molecular data and external morphological characteristics (Tillie et al. 2000; Wilson 2004; Goldblatt and Mabberley 2005; Kang et al. 2020). Goldblatt and Mabberley even renamed the Belamcanda chinensis to Iris domestica (Goldblatt and Mabberley 2005). In the matK phylogenetic tree (Fig. 3), Belamcanda chinensis was embedded within the genus Iris and formed a single clade with I. dichotoma, indicating a closet relatives between the two. We suggested that Belamcanda chinensis be included in the Subgen. Pardanthopsis within the genus Iris.

Research has found that the ITS sequence has many variation sites, providing more genetic information with insertions and deletions. The main variations are located in the ITS1 and ITS2 regions. However, the matK sequence is much more conserved, with variation sites primarily involving single nucleotide changes and occasional insertions or deletions in some species, with fragments generally being only a few base pairs long. Based on current research results, both sequences are effective for classifying Iris species. However, considering sequence features, clustering patterns and support rates, we found that matK is more suitable for studying phylogenetic relationships between subgenera, while the ITS sequence is more suitable for investigating intra-species and inter-species phylogenetic relationships due to its significant variation within the same species from different sources.

Funding

The authors would like to thank the National Natural Science Foundation of China[Grant numbers 22206140].

Data availability

Data will be made available on request.

Competing Interests

The authors declare that they have no conflicts of interest.

Author contributions

Mao-Lin Chen: Writing – original draft, Data curation. Yi-Mei Feng: Data curation. Xin-Yu Zhang: Data curation. Fan Xu: Data curation. Qing-Qing Kang: Data curation. Xin-Jing Ning: Software. Xian-Tong Wang: Software. Xue Xiao: Investigation. Li-Juan Yang: Data curation. Xiao-Fang Yu: Writing – review & editing, Funding acquisition.

Ethical standards

The experiments were performed according to the current laws of China.

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No competing interests reported.

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Phylogenetic study of Iris plants in China based on chloroplast matK gene and nuclear ITS gene

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Introduction

Materials and methods

Plant materials

DNA amplification and sequencing

Phylogenetic data analysis

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Discussion

Classification of Belamcanda chinensis

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