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Research Article
Cardamine tangutorum O.E.Schulz (Brassicaceae), a new synonym of Cardamine macrophylla Willd
expand article infoJia-lu Li‡, Yi He‡, Quan-ru Liu‡
‡ Beijing Normal University, Beijing, China

Corresponding author: Quan-ru Liu ( liuquanru@bnu.edu.cn )

Academic editor: Peter de Lange
© 2025 Jia-lu Li, Yi He, Quan-ru Liu.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Li J, He Y, Liu Q (2025) Cardamine tangutorum O.E.Schulz (Brassicaceae), a new synonym of Cardamine macrophylla Willd. PhytoKeys 255: 123-141. https://doi.org/10.3897/phytokeys.255.146179
Open Access

Abstract

Cardamine macrophylla Willd. and Cardamine tangutorum O.E.Schulz are both widely distributed plants of the genus Cardamine L. in East Asia, and they are regarded as the same species in Flora of China. In this study, through literature and specimen research, morphological measurement and phylogenetic analyses, the results show that the traditional distinguishing characters cannot distinguish the two species. Cardamine tangutorum O.E.Schulz inserted into the branch of Cardamine macrophylla Willd. in the molecular phylogenetic tree. Therefore, based on the results of this study, Cardamine tangutorum O.E.Schulz was treated as the synonym of Cardamine macrophylla Willd.

Key words:

Cardamine, China, morphology, phylogenetics, taxonomy

Introduction

The genus Cardamine L. belongs to the tribe Cardamineae within the Brassicaceae family. It is a cosmopolitan group with approximately 280 species distributed worldwide (Marhold et al. 2021). As one of the global centers of diversity, Flora of China records 48 species of Cardamine L. in China, 24 of which are endemic. New species and distribution records of Cardamine L. in China continue to be reported (Al-Shehbaz 2015a, 2015b; Diao et al. 2023)(Marhold et al. 2007; Al-Shehbaz and Boufford 2008; Chen et al. 2011; An et al. 2016; Marhold et al. 2016; Šlenker et al. 2018; Wu et al. 2021; Guo et al. 2024; Li et al. 2024). To date, there are 61 Cardamine L. species in China, 31 of which are endemic.

In 1753, Linnaeus published the genus Cardamine L. in Species Plantarum, which included 15 plant species. The type species of the genus is Cardamine pratensis L. Additionally, Linnaeus also established the genus Dentaria L. (Linnaeus 1753). In 1769, Crantz merged Dentaria L. into Cardamine L. (Crantz 1769). O. E. Schulz classified Cardamine L. into 13 sections based on the presence or absence of scales on the rhizome surface, ovule number, funiculus morphology, and the relative position of cotyledons and radicles (Schulz 1903)(Schulz 1931, 1936). This system was adopted by major floras such as FRPS and Flora of the USSR (Komarov 1939; Zhou. et al. 1987). In FRPS, Chinese Cardamine L. species were divided into seven sections based on Schulz’s classification system, with most species assigned to the section Cardamine L.

Al-Shehbaz proposed the concept of a broadly circumscribed Cardamine L., defining the genus as a cruciferous group characterized by “flattened and winged seeds, and dehiscent siliques that split elastically through torsion.” This treatment incorporates Dentaria into the expanded Cardamine s. l. and abolishes infrageneric classifications (Al-Shehbaz 1988, 2000, 2015c) Al-Shehbaz and Guang 1998; Zhou et al. 2001). These taxonomic revisions have subsequently received support from molecular phylogenetic evidence (Franzke et al. 1998; Sweeney and Price 2000). Major floristic works, including Flora of China and Flora of Pan-Himalaya, have adopted Al-Shehbaz’s taxonomic treatment of Cardamine L. (Zhou et al. 2001; Al-Shehbaz 2015c).

During the nomenclatural verification and taxonomic studies of Cardamine L. species in China, we found that the identification of specimens for the widely distributed Cardamine macrophylla and Cardamine tangutorum in East Asia was inconsistent, leading to controversies in their species delimitation. Flora of China suggested that these two taxa might represent the same species, requiring further verification (Zhou et al. 2001).

Cardamine macrophylla Willd. was first described by Willdenow, based on specimens collected from the Taz River estuary in the Mangazeya region of northern Siberia, USSR (as shown in Fig. 2). The distribution of Cardamine macrophylla spans a wide latitudinal range, occurring in Siberia, the Qinling Mountains, and the Hengduan Mountains. Due to its significant morphological variation, numerous subspecies and varieties have been described under this species. In 1825, D. Don described Cardamine polyphylla D. Don (Don and Hamilton 1825). In 1872, several varieties of Cardamine macrophylla were published in Flora of British India: Cardamine macrophylla var. dentariifolia Hook. f.& T. Anderson, Cardamine macrophylla var. lobata Hook. f.& T. Anderson, and Cardamine macrophylla var. sikkimensis Hook. f.& T. Anderson. Additionally, the nomen nudum Cardamine foliosa Wall. was treated as Cardamine macrophylla var. foliosa Hook. f.& T. Anderson (Wallich and East 1828; Hooker 1875). In Schulz’s 1903 system, Cardamine macrophylla was placed in the Macrophyllum section (Schulz 1903).

In 1980, Tai Yien Cheo and colleagues treated Cardamine polyphylla D. Don as a variety of Cardamine macrophylla Willd., naming it Cardamine macrophylla Willd. var. polyphylla (D. Don) T. Y. Cheo et Fang. They also described a new variety, Cardamine macrophylla Willd. var. diplodonta T. Y. Cheo, based on variations in leaf margin serration and the number of cauline leaves (Cheo et al. 1980). In the FRPS, three varieties of C. macrophylla were recognized: C. macrophylla Willd. var. polyphylla (D. Don) T. Y. Cheo et Fang, C. macrophylla Willd. var. diplodonta T. Y. Cheo, and Cardamine macrophylla var. crenata Trautv. (Trautvetter 1887). However, the 2001 edition of Flora of China synonymized these varieties under C. macrophylla. Additionally, it synonymized Dentaria sino-manshurica Kitag (Kitagawa 1937) and Cardamine urbaniana O.E. Schulz (Schulz 1903) with C. macrophylla (Zhou et al. 2001).

Cardamine tangutorum O.E. Schulz was described by Schulz in 1903. According to the protologue, this species is distinguished by its purplish-red, prominently veined sepals with translucent narrow margins and its triangularly swollen ovule stalks measuring 0.5–4.0 mm in length. Schulz cited multiple specimens as syntypes for this species: Gansu 1872 N. M. Przewalski #s.n.,1879 N. M. Przewalski #s.n.,1880 N. M. Przewalski #s.n.; Gansu 1885 G. N. Potanin #s.n.; Shaanxi, Miaowang Mountain 1899 J. Giraldi No. 3379 No. 3378 ;Shaanxi 1884 Potanin #s.n.; Beijing Xiaowutai Mountain 1879 Möllendorff #s.n.; Sichuan 1894 Rosthorn No. 2583 (see Fig. 1). In Schulz’s classification system for Cardamine L., C. tangutorum was placed in the section Dentaria (Schulz 1903). Both the FRPS and Flora of China recognized C. tangutorum. However, the editors of Flora of China noted the morphological similarity between C. tangutorum and C. macrophylla, suggesting that they might represent the same species (Zhou. et al. 1987; Zhou et al. 2001), with a common species distributed in subalpine and alpine zones in the Himalayas, North China, Northeast China, Central China, and Hengduan Mountains.

Figure 1. 

Syntype specimens of Cardamine tangutorum including 1880 N. M. Przewalski #s.n. (top left), 1885 G. N. Potanin #s.n. (top right), 1879 N. M. Przewalski #s.n. (bottom left), and Rosthorn #2583 (bottom right).

Figure 2. 

Holotype specimen of Cardamine macrophylla.

According to records from Flora Reipublicae Popularis Sinicae, Flora of China, and Flora of Pan-Himalaya, the morphological distinctions between Cardamine macrophylla and C. tangutorum are primarily as follows: C. macrophylla exhibits a conspicuously thickened rhizome lacking scale-like appendages, a plant height of (20–) 30–95 (–115) cm, 3–12 cauline leaves scattered along the stem, and uppermost leaflets of the cauline leaves decurrent into winged extensions. C. tangutorum is characterized by a slender, whip-like rhizome with scale-like appendages, a plant height of (8–) 15–30 (–40) cm, 1–3 cauline leaves clustered at the upper stem, and comparatively narrower and smaller cauline leaves with fewer lateral leaflets and non-decurrent uppermost leaflets.

Based on the above understanding, we conducted morphological and molecular phylogenetic studies, supported by a thorough review of the literature and examination of specimens, to clarify the taxonomic status of these two species.

Method

Sampling and morphological analyses

Specimens with complete rhizoid and multiple copies were selected from PE, BNU, KUN and other herbaria, as well as the digital HD and other type specimen photos on the website of LE Herbarium. Specimens’ collection are shown in Table 1. A total of 46 population specimens were collected for morphological analyses. Re-identified and grouped according to the retrieval characters described above. Ten traits were selected according to FRPS, Flora of China, and protologue, and measured by ImageJ (Schindelin et al. 2012), and two ratios were calculated. The characters and coding are listed in Table 2. PCA was performed by Past 4.13 after standardization by IBM SPSS Statistics (v.27) (Hammer et al. 2022; IBMCorp 2020).

Table 1.
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Taxa and sample sites, bold specimen representation was used for molecular phylogenetic analysis.

Taxa and sample sites
C. tangutorum O.E. Schulz
China Sichuan, Zhongsu Team, 7165
China Sichuan: Wenchuan, K.Y.Lang ,L.Q.Li, Y. Fei, 1136
China Sichuan: Aba, K.Y.Lang ,L.Q.Li, Y. Fei, 2048
China Qinghai: K.M.Liou, 6185
China Qinghai, Z.H.Zhang etc, 4484
China Gansu, T.P.Wang 7275
China Gansu, T.P.Wang 6979
China Gansu, Tsi-Tang Li198
China Hebei: Tuoliang, TL006
China Hebei: Tuoliang, TL008
China Hebei: Tuoliang, TL023
China Hebei: Tuoliang, TL007
China Beijing: Donglingshan, C. Wang, 50611006
China: Eastern Gansu, G. H. Potani, 1885
China Gansu: Tanggulashan, N. M. Przewalski, 1872
China Hebei: Xiaowutaishan, 62079 CC. W. Wang
China Gansu: 1879, N. M. Przewalski #s.n. (syntype of C. tangutorum)
China Gansu: 1880, N. M. Przewalski #s.n. (syntype of C. tangutorum)
C. macrophylla Willd.
China Chongqing, Chengkou, G212 Road, zhang698
China Sichuan, Aba, zhang801
China Sichuan: Bazhong, GSL2015050209
China Hunan, longshan, Bamianshan, zhang667
China Hubei, Yanzi Zhen, zhang676
China Yunnan, T.T.yu 9780
China Sichuan, S.X, Yu, Y.T.Hou, X.X.Zhang, Y.M.Zhao, 4812
China Sichuan, K.Y.Lang, L.Q.Li, Y.Li, 1917
China Sichuan: Batang, K.Y.Lang, L.Q.Li, Y.Li, 2471
China Sichuan: Nanping, K.Y.Lang, L.Q.Li, Y.Li, 1608
China Hebei: Xilingshan, J.X.Duan, 239
China Shanxi: Nuanshuihe, Shanxi Investigation Team, 530
China Shanxi: Shiziping, Shanxi Investigation Team, 592
China Shanxi: Yuwu, J.M.Liu, 1662
China Henan: Neixiang, D.E.Boufford,C.Y.Xi,T.S.Ying etc.26303
China Hubei: Shennongjia, Hubei Shennongjia Planting Research Team, 10006
China Anhui: Yuexi, X.L.Liu, 492
China Hunan: Sangzhi, B. Zhang & X. Xiang, 090425022
China Sichuan: Kangding, Chuanxi Team, K.J.Guan, W.C.Wang etc,772
China Sichuan: Kangding, Chuanxi Team, K.J.Guan, W.C.Wang etc,387
China Sichuan: Kangding, Y.T.Zhang & K.Y.Lang, 37
China Sichuan: Ganzi, Xizang Team, 73-03
China Sichuan: Ganzi, Y.T.Zhang & K.Y.Lang, 121
China Sichuan, E.H.Li,Y.F.Han, J.G.Liao, Y. Hu, H82-335
China Sichuan: Muli, Qingzang Team, 13042
China Sichuan: Xiangcheng, Qingzang Team & Hengduanshan Team, 003712
China Sichuan: Yanyuan, Qingzang Team, 12344
China Sichuan: Maerkang, X. Li, 70477
China Sichuan: Jiulong, Z.X.Tang, X.W.Tian, Q.G.Sun, 245
Table 2.
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List of morphological characters and their acronyms used in analyses.

Acronym Description of character
Scaly* Presence of scales on rhizome (with 1/without 0)
RW Rhizome diameter
CW Mid-stem diameter
H* Height of stem
LH* Height of the location of lowest stem leaves
No. SL* Number of stem leaves
No. MLL* Number of lateral leaflets on mid-stem leaves
LTL* Length of the terminal leaflet on mid-stem leaves
WTL Width of the terminal leaflet on mid-stem leaves
DL* Length of the uppermost leaflet’s lower edge on mid-stem leaves
Ratio
LH/H*,LTL/WTL*,RW/CW*

Phylogenetic analyses

Fresh plant leaves were collected in the field and quickly dried with silica gel. Plant samples were sent to Beijing Novogene Corporation for quality testing and re-sequencing. The sequencing platform, Illumina HiSeq X Ten and BGI, was used to generate approximately 6 GB of data for each sample. The chloroplast genome was assembled from the clean data using Get Organelle (Jin et al. 2020). Plastid Genome Annotator (PGA) was used to annotate the chloroplast genome with Amborella trichopoda Baill. from software as references (Qu et al. 2019). Then, 26 plastid genome sequences were downloaded from NCBI (Table 7), including 23 species of Cardamine L.and 2 species, Rorippa sylvestris (L.) Besser, Rorippa indica (L.) Hiern as outgroup. The annotated sequences were imported into PhyloSuite (Zhang et al. 2020), the Mafft module was used for sequence alignment (Katoh et al. 2019), and the ModelFinder module was used to calculate the nucleotide substitution model for the aligned sequences. The maximum likelihood (ML) tree was constructed using IQ-TREE (Minh et al. 2020), with the nucleotide substitution model set to GTR+R3+F and a standard bootstrap value of 1000.

Flow cytometric measurements and estimation of DNA ploidy levels

Methods referring to Marhold et al. (2010) and Kobrlová and Hroneš (2019) measured the nuclear DNA content using flow cytometry. Inferred the DNA ploidy levels within the studied populations based on Cardamine L.species with known ploidy. The relative nuclear DNA content was determined using PI, a DNA intercalating fluorescent dye, with arbitrary units (a.u.) as the unit of measurement. The buffer solution used was LB01. Dehydrated leaves, preserved by drying at 40 °C for 18–24 months, were used for the determination of chromosome ploidy. The sample sources and voucher specimens are presented in Table 3. In a pre-cooled culture dish, 1–2 mL of LB01 buffer solution and 2 cm2 of dry leaves were added. After rapid chopping, the mixture was filtered through a 400-mesh gauze, centrifuged at 4 °C, 3000 rpm for 10 minutes, and the supernatant was discarded. The pellet was re-suspended in 600 μL of LB01 buffer solution, followed by the addition of 100 μL of PI solution (50 μg/mL), and stained in the dark for 15 minutes. Ploidy level of the stained cell suspension was determined by flow cytometry (ACEA NovoCyte 3130). Using 488 nm blue light excitation, 10,000 cells were collected at a time. The other samples were determined under the same voltage bar using C. scutata (2n = 4x = 32) as the reference for tetraploid.

Table 3.
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List of taxa and sample sites used in flow cytometric measurements.

Taxa Voucher information
C. tangutorum BNU2023WLH077
BNU2022xz
BNU2023ZJK26
BNU2022HLG 002
C. macrophylla BNU2022YN070
BNU2022YN004
BNU2022mcs006
BNU2022em002

Result

Principal component analysis results

Using SPSS software, a cluster analysis was conducted on the standardized data matrix, resulting in a character correlation matrix as shown in Table 4. The cluster analysis results indicate that none of the Pearson correlation coefficients between each pair of characters reached 0.8. The highest correlation was between the length of the apical leaflet of the stem leaves and the downwards extension length of the first pair of lateral leaflets, with a coefficient of 0.753. Thus, no significant correlations were observed among other traits, allowing all traits to be retained for subsequent multivariate principal component analysis.

Table 4.
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Correlation of morphological characters between C. macrophylla and C. tangutorum.

H LHH No. SL No. MLL LTL LTL/WTL DL RW/CW SCALY
H – -0.16 0.209 0.402 0.597 0.209 0.509 0.295 0.037
LHH – -0.653 -0.136 -0.161 0 -0.08 -0.05 0.041
No. SL – 0.114 0.162 0.171 0.201 0.049 -0.052
No. MLL – 0.298 0.093 0.312 0.16 0.002
LTL – 0.267 0.753 0.431 0.271
LTL/WTL – 0.325 0.102 -0.03
DL – 0.37 0.045
RW/CW – 0.119
SCALY –

Principal component analysis (PCA) was performed on the standardized data matrix using PAST software, and the contribution values of each principal component (PC) are shown in Table 5. The cumulative contribution of the first four principal components was 77.658%, indicating successful dimensionality reduction, making PCA suitable for this study. Based on previously identified results, the data was grouped and incorporated into the standardized data matrix, as shown in Fig. 3.

Table 5.
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Contribution Values of Each Principal Component.

Principal component (PC) Eigenvalue Proportion (%) Cumulative (%)
PC1 2.938 36.615 36.615
PC2 1.511 18.828 55.443
PC3 0.956 11.919 67.362
PC4 0.826 10.296 77.658
PC5 0.601 7.493 85.151
PC6 0.475 5.917 91.067
PC7 0.351 4.368 95.435
PC8 0.244 3.0354 98.471
PC9 0.123 1.5291 100.000
Figure 3. 

Ordination diagrams of principal component analyses, Grouping reference specimen identification.

Based on the results of the PCA, the distribution ranges of C. macrophylla and C. tangutorum along the PC3 and PC2 axes are essentially identical. Along the PC1 axis, the two species exhibit a clear continuous transitional distribution, with significant overlap in their primary distribution ranges and their core distribution areas also show substantial overlap. In PC1, characteristics with larger contributions include the length of the downwards extension of the uppermost lateral leaflets of the stem leaves, the length of the apical leaflets of the stem leaves, and plant height. In PC2, the characteristic with the largest contribution is the position of leaf attachment. For PC3, the characteristics with the highest contributions are the leaf length-to-width ratio and the number of lateral leaflets on the stem leaves. Box plots of these high-contributing traits, as shown in Fig. 4, indicate that the distribution ranges of all these high-contributing characteristics also demonstrate significant overlap.

Figure 4. 

Box Plots of Traits with High Principal Component Contributions.

Molecular phylogenetic study

The chloroplast genome tree is illustrated in Fig. 5, with bootstrap support (BS) values indicated below the branches. The Cardamine L. species selected for this study form a well-supported monophyletic group, divided into three distinct clades. C. tangutorum is nested within the monophyletic group formed by C. macrophylla, both clustering together in clade 3. Besides C. macrophylla and C. tangutorum, morphologically similar species such as C. leucantha and C. fragarifolia were included in constructing the phylogenetic tree.C. leucantha clusters with C. impatiens at the base of clade 3. C. leucantha forms a monophyletic group with C. glanduligera, positioned within clade 3.

Figure 5. 

The strict consensus tree resulted from IQ-TREE analysis using plastid genome sequence. Bootstrap values (BS) are shown under branches. Red marking C. macrophylla, blue marking C. tangutorum.

Flow cytometric measurements and estimations of relative DNA content

The relative DNA content between different populations of C. macrophylla and C. tangutorum was calculated using 12-to 24-month-old specimens. The results showed that for all the populations measured in this study, the coefficient of variation ranged from 0.49% to 6.89%, as shown in the Table 6, indicating that the results were credible and that the relative DNA content of C. macrophylla and C. tangutorum was stable within the populations. The relative DNA content of C. macrophylla and C. tangutorum is shown below; the relative DNA content of C. macrophylla and C. tangutorum varies greatly among populations, but the distribution range at the species level is basically the same (shown in Fig. 6).

Table 6.
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Relative genome sizes obtained for C. macrophylla, C. tangutorum.

Taxa Voucher information Relative genome size in a.u. (arbitrary units) Variation (%)
C. tangutorum BNU2023WLH077 1.316 1.89%
C. tangutorum BNU2022xz 1.018 6.49%
C. tangutorum BNU2023ZJK26 1.415 6.13%
C. macrophylla BNU2022YN070 1.440 3.10%
C. macrophylla BNU2022YN004 0.888 6.02%
C. macrophylla BNU2022mcs006 1.333 4.33%
C. macrophylla BNU2022em002 0.985 5.45%
C. tangutorum BNU2022HLG 002 1.426 0.49%
Table 7.
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Taxa in phylogenetic analyses from GenBank.

GenBank accession numbers Species name
NC026446 C. resedifolia
MT136871 C. quinquefolia
MK637691 C. pentaphyllos
NC036964 C. parviflora
NC036963 C. oligosperma
MZ043777 C. occulta
MF405340 C. macrophylla
MZ846206 C. lyrata
MK637684 C. kitaibelii
NC026445 C. impatiens
ON322745 C. hupingshanensis
MK637681 C. hirsuta
MN651504 C. heptaphylla
MK637680 C. glanduligera
MZ043778 C. fallax
NC049605 C. enneaphyllos
OL634846 C. circaeoides
NC049603 C. bulbifera
MN651509 C. bipinnata
MZ043776 C. amariformis
NC036962 C. amara
NC060863 C. abchasica
KJ136821 C. impatiens
NC069649 Rorippa sylvestris
NC065833 Rorippa indica
Figure 6. 

Relative genome sizes obtained for C. macrophylla, C. tangutorum.

Discussion

Taxonomic status of C. tangutorum

Through morphological analysis of 46 populations, it was found that the distinguishing features between C. macrophylla and C. tangutorum significantly overlap. The decurrence in leaflets of C. macrophylla and C. tangutorum significantly overlaps in their distribution ranges. Generally, the decurrence in leaflets of C. macrophylla tends to be longer. However, some populations, such as Qingzang Expedition 12344 and T.T.yu 9780, exhibit nearly no decurrence in leaflets. Other related species within the genus, such as C. lyrata and C. occulta, exhibit continuous variability in decurrence in leaflets, making this trait unsuitable as a basis for species differentiation. The position of stem leaves is also a significant distinguishing feature in FRPS (Zhou. et al. 1987), but as shown in the boxplot, the lower stem leaf positions of both C. macrophylla and C. tangutorum are essentially the same, located in the upper part of the plant, cannot be used for distinguishing.

Previously, when C. tangutorum was described, Schulz cited many collections as syntype specimens. Comparing the syntype specimens of C. tangutorum with the holotype specimens of C. macrophylla, decurrence in leaflets cannot be observed at the leaflet bases in C. macrophylla holotypes. Furthermore, significant variations in leaf length-width ratios, sizes, and positions were observed between different isotype specimens of C. tangutorum (as shown in Fig. 1).

Upon examining the extant specimens of C. macrophylla and C. tangutorum in herbariums, we found that for both species, individuals with slender whip-like rhizomes have grooves on the rhizome surface but lack significant scales. However, some grayish-white triangular scales or leftover marks after their detachment were commonly present at the bases of leaf buds and branch buds (as shown in Fig. 7).

Figure 7. 

Rhizomes of C. tangutorum and C. macrophylla (A, B C. tangutorum C–F C. macrophylla). Scale bar: 1 cm.

Considering that both C. macrophylla and C. tangutorum are perennial herbaceous plants with persistent rhizomes, these scaly appendages should be regarded as bud scales. In individuals with significantly fleshy rhizomes, these scales on the rhizome surface are less prominent. The presence or absence of scales on rhizomes, as emphasized in Schulz’s classification system, was considered an important criterion for subgroup classification. In Schulz’s system, a key feature of the Cardamine L. group is the prominent scales on rhizomes, whereas the macrophylla group lacks them. However, in The Families and Genera of Angiosperms in China it was suggested that the two groups in China should be merged, negating Schulz’s classification viewpoint (Wu et al. 2003). Examining the specimens, it is evident that the prominence of rhizome scales is affected by the plant’s age and the degree of rhizome fleshiness and does not serve as a basis for species differentiation. The quantitative taxonomy results indicate that none of the traditional differentiating traits effectively distinguish C. macrophylla from C. tangutorum. C. macrophylla shows significant morphological variability, within which C. tangutorum should be included.

Some species of Cardamine L., such as C. yezoensis, C. pratensis, etc., have variation in ploidy and relative DNA content within species (Marhold et al. 2010). The results of flow cytometry showed that the relative DNA content of C. macrophylla and C. tangutorum was stable within populations, but there was great variation among populations, showing polymorphism of ploidy and relative DNA content. However, the distribution range of DNA content of C. macrophylla and C. tangutorum were basically the same, which supported the combination of C. macrophylla and C. tangutorum.

Multiple populations of C. macrophylla and C. tangutorum were selected for the molecular phylogenetic study. The results indicate that both species share a high similarity in chloroplast genomes, with C. tangutorum nested within the monophyletic clade of C. macrophylla. This suggests close phylogenetic relationships at the molecular level, making it inappropriate to treat them as distinct species. C. leucantha and C. fragarifolia, with similar leaflet morphology to both C. macrophylla and C. tangutorum, all with long lanceolate leaflets and cuneate bases, are positioned within clade 3 in the molecular phylogenetic tree. This possibly suggests the single evolutionary origin of these morphological characters.

Combining molecular phylogenetic, cytological and morphological studies, it is concluded that the morphological range of C. macrophylla and C. tangutorum significantly overlaps, making them difficult to distinguish. Their molecular phylogenetic positions are nested within the same monophyletic group, rendering them indistinct. Considering the common species distributed, C. macrophylla and C. tangutorum should be treated as a single species, and C. tangutorum should be treated as a synonym of C. macrophylla.

Taxonomic treatment

Cardamine macrophylla Willd., Sp. Pl. 3 (1): 484. 1800.

=Cardamine macrophylla var. crenata Trautv., Trudy Imp. S.-Peterburgsk. Bot. Sada 5 (1): 18. 1877.

=Cardamine macrophylla var. dentariifolia Hook. f.& T. Anderson Fl. Brit. India [J. D. Hooker] 1 (1): 139 (1872). Type (designated by (Marhold et al. 2015)): India, Himal.Bor.Occ., Thomson, T. s.n., (K000397478!)

=Cardamine macrophylla var. diplodonta T.Y. Cheo, Bull. Bot. Lab. N.-E. Forest. Inst., Harbin 6: 20. 1980.

=Cardamine foliosa Wall.,[Numer. List: 4779. 1831. nom. nud.]

≡Cardamine macrophylla var. foliosa Hook. f.& T. Anderson Fl. Brit. India [J. D. Hooker] 1 (1): 139 (1872). Type(designated by (Marhold et al. 2015)): India, Kumaon, Wallich, Cat.Wall.4779 (K000247365!, P00747537!, B_10_0241370!, B_10_0241369!, GH00549127!).

=Cardamine macrophylla var. lobata Hook. f.& T. Anderson, Fl. Brit. India [J. D. Hooker] 1 (1): 139 (1872). Type(designated by (Marhold et al. 2015)): [INDIA] [Label 1]: “marshy meadows, Nira Zanskar, 12,900 ft, 2 July 1849 [?]”, [Label 2]: “Hab. Himal. Bor. Occ., W. Tibet, Regio Temp., Alt. 12,900 ft, T. T. [T. Thomson] s.n.” (K000397477).

=Cardamine macrophylla var. moupinensis Franch., Pl. David. 2: 18. 1888. Type(designated by (Marhold et al. 2015)): [CHINA], [Label 1 (handwritten)]: “Moupin, Thibet oriental, lieux frais en montagne, Avril 1869”, [Label 2 (printed)] “Chine (Thibet Oriental), Province de Moupin, 1870 [sic!], David s.n.” (P00747519, Isolectotype: P00747518).

=Cardamine macrophylla var. sikkimensis Hook. f.& T. Anderson Fl. Brit. India [J. D. Hooker] 1 (1): 139 (1872). Type (designed by (Marhold et al. 2015)): India, Sikkim Lachung, 03 September 1849, Hooker, J.D. s.n. (K000397479!, isolectotype: K000397480!, K000397481!, GH00549128!) =Cardamine polyphylla D. Don not O. E. Schulz, Prodr. Fl. Nepal.: 201. 1825.

=Cardamine sachalinensis Miyabe & T. Miyake, Fl. Saghalin No. 58, t. 3, fig. 1–3, 1915.

=Cardamine sino-manshurica Kitag., Rep. Inst. Sci. Res. Manchoukuo 4: 111, 1940.

≡Dentaria sino-manshurica Kitag.Rep. Inst. Sci. Res. Manchoukuo 4: 111, 1940.

=Cardamine urbaniana O.E. Schulz, Bot. Jahrb. Syst. 32 (2–3): 396. 1903.

Syntype: China, Sichuan, 1885–1888, A. Henry 5635 (B 10 0241328!); China, Shaanxi, Huangcaoping County, 20 June 1894, G. Giraldi 447 (B 10 0241329!)

=Dentaria gmelinii Tausch, Flora 19 (2): 402, 1836.

=Dentaria macrophylla Bunge ex Maxim., Prim. Fl. Amur. 45, 1859.

=Dentaria wallichii G. Don, Gen. Hist. 1: 172, 1831.

=Dentaria willdenowii Tausch, Flora 19 (2): 403, 1836.

=Cardamine tangutorum O.E. Schulz, Bot. Jahrb. Syst. 32 (2–3): 360. 1903.

Holotype.

Russia, Northern Sibiria, Mangezeya, at the mouth of Taz River (B-W11970-030!)

Syntype.

China, Gansu, Terra Tangurorum, N. M. Przewalski No. 1872 (LE01014556!); China, Gansu, Terra Tangurorum, N. M. Przewalski No. 1873 (K000697742!); China, Gansu, Terra Tangurorum, N. M. Przewalski No. 1880 (LE01014557!); China, Gansu, orient, G. N. Potanin 1885 (LE01014555!, LE01014558!); China, Sichuan, “Tsakulao”,1891, A. v. Rosthorn 2583., (B 10 0241334!); China, Shaanxi, Baoji County, Mountain Miaowangshan, 1899, J. Giraldi No. 3379; China, Hebei, Mountain Xiaowutaishan, 1879, O. V. Möllendorff s. n.

Habitat.

Often in shady areas under forests, along ditches or in subalpine meadows.

Distribution.

Widely distributed in Siberia, Mongolia, Himalayas, North China, Northeast China, Central China, Hengduan Mountains.

Phenological period.

Flowering from May to July, fruit from June to September.

Description.

Perennial herbs, 30 cm–70 cm tall, up to 1 m. Rhizomes creeping, sometimes tuberous; basal leaves pinnate, pedicled on creeping rhizomes, terminal leaflet long ovate to lanceolate, margin toothed, lateral leaflet similar to terminal leaflet. Cauline leaves are similar to basal leaves but slightly smaller, mostly in the middle and upper parts of the plant. Flowers lilac to purplish red, calyx margins white membranous, petals with long claws; the seeds are oblong. This species showed high diversity in the size of leaflets and morphology of leaf margins.

Specimens examined.

• Sichuan Province: Yajiang County, Mountain Kazila, 08 September 2011, He et al. SCU-11-360, (KUN1235449!, KUN1235450!); Kangding County, He et al. SCU-080340, (KUN1235446!); • Yunnan Province: Shangri-La County, 24 July 2014, Guo et al. 14CS9432, (KUN1321027!); • Heilongjiang Province: Jinshantun County, Huilongwan National Forrest Park, Hou et al. 389, (QFNU0006577!, QFNU0006578!); • Hebei: Donggou Temple, 16 May 1951, Wencai Wang 2122, (PE 01004424!, PE 01004426!); Xiaowutai Mountain, Tielin Temple, 29 July 1906, Y. Yabe s.n., (NAS00326894!, NAS00326895!) • Shanxi Province: Mountain Guandishan, 23 June 1959, Sai Ma 15030, (WUK0321706!, WUK0324493!); Wutaishan Mountain, 17 July 1907, Y. Yabe s. n., (NAS00326872); Lingchuan County, Fenghuang Valley, 14 April 2014, Kong et al. k0087, (SD00018202!, SD00016344!) • Tibet Province: Mangkang County, 318 Road, 20 July 2008, Zhang et al. SunH-07ZX-0503 (KUN1300753!, KUN1300754!, KUN1300755!); Yadong County, Naiduila Mountain, 23 August 2013, Y.S.Chen et al.13-1966, (PE02000587!, PE02000588!); Dingqing County, 22 July 2016, Shuai Li et al. 20167324, (BNU0030842!); • Gansu Province: Tan County, Mountain Lougu, 15 June 1956, Huanghe Exped 4911, (WUK0085438!); Zhuoni County, Chebagou Valley, Yin et al. LiuJQ-GN-2011-128, (KUN1235443!); Maqu County, Xiuma, 03 September 2008, Li et al. LiJ0061, (KUN1235442!); Maqu County, Langmu Temple, 01 June 1999, Bailong River Exped 1589, (PE01556040!); Lianhua Mountain, 13 May 2002, Xuegang Sun 2741, (PE01998146!); Linxia County, Dalijia Mountain, 28 July 1988, Ji Ma 88090, (NAS00326893!, NAS00326896!, NAS00326897!); • Beijing: Baihua Mountain, August 1981, Anonymous 81-0322, (BJFC00019955!, BJFC00019954!, BJFC00019953!); Baihua Mountain, June 1990, D. D. Lu et al. 272, (BJFC00019961!, BJFC00019960!, BJFC00019959!, BJFC00019956!); Mentougou County, Lingshan Mountain, 01 May 2009, G. M. Zhang 200923, (BJFC00066271!, BJFC00066273, BJFC00066274!); • Shaanxi: Long County, 19 May 1983, Sujia River, J. X. Yang 4207, (WUK0438580!, WUK0438581!); • Qinghai Province: Ledu County, Qutan, 24 June 1970, Benzhao Gou 7198, (WUK0308377); Menyuan County, 26 July 2008, Yuhu Wu LJQ-QLS-2008-0143, (KUN1235444!, KUN1235445!); Maqin County, Dawu, 14 July 2014, Xiaoyu Wu, Xiaolei Zhang s.n., QH2014006, (BNU0020082!); Huzhu County, Beishan Foresty Centre, 13 July 1982, B. Z. Gou 25521, (HNWP102005!); Guide County, Laji Mountain, 20 June 1992, R. F. Huang 3710, (HNWP169550!, HNWP169551!); • Mongolia: 17 August 1979, Губанов 7845, (MW0179858!, MW0179859!);17 August 1979, Губанов 7830, (MW0179855!, MW0179856!); 14 August 1979, Губанов 7704, (MW0179853!, MW0179854!); 9 August 1979, Губанов 7610,(MW0179857!, MW0179863!); • Russia, Altai & Sayany Mountains: 9 July 1984, Триль 4493, (MW0081495!); 27 July 1983, Шауло 2019, (MW0081478!); 17 July 1984, Сонникова 1535, (MW0081469!);18 June 1983, Сонникова 1532, (MW0081472!); 1 August 1982, Сонникова 1529, (MW0081468!); 8 August 1979, Ухина 1518, (MW0081474!); 25 June 1978, Ухина 1515, (MW0081471!); 13 June 1988, Шауло 30, (MW0081475!, MW0081494!); • Russian Far East: 16 August 1990, Штрик 90-569, (MW0081357!, MW0081364!); 23 August 1990, Штрик 90-449, (MW0081358!, MW0081372!); 8 August 1990, Борисов 90-189, (MW0081369!); 28 July 1988, Кемниц 88-238, (MW0081367!); 25 July 1998, Кемниц 88-222, (MW0081366!); • Central Siberia: 23 July 1977, Куваев 85,(MW0081457!) • Baikal & Transbaikal region: 9, August 1929, Назаров 12.819, (MW0081409!); 7 July 1929, Назаров 12.118, (MW0081404!) • Kazakhstan, Western Altai Mountains: 27, August, 1932, Воронов 720, (MW0081463!); 18 July 1930, Смирнов 173, (MW0081461!, MW0081462!).

Acknowledgments

We would like to express gratitude to the staff of PE, KUN for their support of this study.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This research was financed by the National Natural Science Foundation of China (No. 210100203 & No. 32200170).

Author contributions

All authors have contributed equally.

Author ORCIDs

Jia-lu Li https://orcid.org/0009-0002-8147-5157

Yi He https://orcid.org/0000-0002-6925-7299

Quan-ru Liu https://orcid.org/0000-0003-4270-4746

Data availability

All of the data that support the findings of this study are available in the main text.

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