Chromosome numbers and karyotypes of 18 species of Epimedium sect. Diphyllon (Berberidaceae) from central China

ABSTRACT

Epimedium is the largest and one of the most important herbaceous genera in Berberidiaceae, especially for section Diphyllon, which has a diversification center in central China. Chromosome numbers and karyomorphology of 24 accessions were examined, representing 18 Epimedium species, five of which were counted here for the first time. A chromosome number of 2n = 2x = 12 was observed for all taxa and all karyotypes were Stebbins’ 2A, except for E. acuminatum, E. brevicornu, E. ilicifolium and one accession of E. leptorrhizum, which were 3A. Their asymmetry index (AI) values vary from 1.71 to 4.56. Intercalary satellites are commonly found on the first or second pair. More than two individuals were sampled for E. franchetii, E. leptorrhizum, E. stellulatum, and E. wushanense and all had consistent and stable cytological traits with respect to their karyotype formula, Stebbins’ asymmetry, and AI values, except for E. leptorrhizum. The significance and limits of the cytological results for Epimedium are also briefly discussed.

KEYWORDS:

• Berberidaceae
• chromosome number
• karyotypes
• Epimedium sect. Diphyllon

1. Introduction

Epimedium Linnaeus (1753), a morphologically diverse genus, is the largest perennial herbaceous genus in Berberidaceae, with more than 50 species widely disjunct between the Mediterranean region and eastern Asia (Stearn 2002; Ying 2002; Zhang et al. 2006). With more than 40 species, China is the most important center of diversity for Epimedium (Ying 2002; He and Xu 2003; Guo et al. 2007, 2008; Zhang and Li 2009; He et al. 2010; Sheng and Tian 2011; Ying et al. 2011; Zhang et al. 2014a, 2015, 2016; Wei et al. 2017). Moreover, all of these species are endemic to China except for E. koreanum Nakai, which is also found in Korea (Ying et al. 2011). Epimedium is well-known as a traditional Chinese medicine that is reported to be effective in strengthening the kidneys, curing rheumatism, and treating osteoporosis, hypertension, and coronary heart disease, as well as strengthening immunity and preventing dementia (Guo and Xiao 1999; Sun et al. 2005). Icariin flavonoids from Epimedium have been shown to inhibit the growth of cancer cells (Zhang et al. 2002; Xie and Sun 2006).

Recent molecular phylogenetic studies have suggested that the genus is differentiated into five groups largely corresponding to the taxonomical classification of two subgenera and four sections (Stearn 2002; Sun et al. 2005; Zhang et al. 2014b). Stearn (2002) treated all Chinese species as subgenus Epimedium section Diphyllon, which is further split into four series based on flower morphology. Although amplified fragment length polymorphism (AFLP) evidence suggests that five well-supported clades are recognized in Epimedium sect. Diphyllon, the backbone relationship of the section remains unclear (Zhang et al. 2014b). Species delimitation in Epimedium has also long been in debate due to diverse morphological traits, especially for the Chinese species of Epimedium sect. Diphyllon, which has wide variations in petal type, form, the relative size of the inner sepals and petals, and flower dimensions (Ying 2002; Zhang et al. 2006, 2015; Smet et al. 2012).

Cytological data, such as chromosome numbers and karyotypes, are very important in understanding plant evolution and diversification (Stebbins 1950, 1971; Hong 1990; Stace 2000), which are significant characters in attempting to deduce phylogenetic relationships in angiosperms (Raven 1975). To date, more than 40 Epimedium taxa have reported chromosome numbers and/or karyotypes (e.g. Tanaka and Takahashi 1981; Takahashi 1989; Sheng and Chen 2007, 2008; Zhang et al. 2008; Sheng et al. 2010; Yan et al. 2016). All examined taxa have somatic chromosome numbers of 2n = 2x = 12, except for E. yingjiangense with a somatic chromosome number of 2n = 4x = 24 (Sheng and Tian 2011). Furthermore, the cyto-morphology of all previously examined taxa closely resemble one another with respect to size and shape of chromosomes, with most characterized as relatively primitive 2A and a few as 1A or 3A (Zhang et al. 2008; Sheng et al. 2010; Yan et al. 2016). A secondary constriction has commonly been found on the proximal side of the short arm of chromosome pair I or II for most species (Sheng and Chen 2008; Sheng et al. 2010).

With the report of many recently discovered Chinese species, the interspecific relationships of Epimedium sect. Diphyllon have become more complex, further confusing the delimitation and systematic arrangement of the species in this genus. The recent interest in understanding the development of Epimedium has prompted the need for in-depth taxonomic and phylogenetic studies of the genus, especially for the highly diverse Chinese taxa in Epimedium sect. Diphyllon. Chromosome numbers and karyotypes have been already reported for a large number of Chinese species from sect. Diphyllon (Sheng and Chen 2007, 2008; Zhang et al. 2008; Sheng et al. 2010; Yan et al. 2016); however, more than one fifth of the Chinese species are lacking chromosome counts. It is necessary to further accumulate cytological data for Epimedium spp. from China. In the present study, we report chromosome numbers and karyotypes from Epimedium sect. Diphyllon species from China and then evaluate possible infrageneric and intergeneric relationships based on our results and previous data.

2. Materials and methods

Cytological examinations were performed on somatic metaphases of root-tips using the squash technique. A total of 24 populations, representing 18 species of Epimedium, were collected between 2016 and 2017 in central China and cultivated in a nursery at Jishou University, Hunan, China. Voucher specimens were deposited in the Herbarium of Jishou University (JIU). Fresh root tips c.1 cm long were cut, pretreated in 1/100 colchicine solution at 22–24°C in the dark for 4 h and then fixed with Carnoy’s fluid (1:3 glacial acetic acid/absolute alcohol) at approximately 4°C for 40 min. The root tips were then placed in 70% ethanol for 10 min and rinsed in distilled water twice for 20 min. Prior to staining, the root tips were hydrolyzed in 1:1 1 N HCl to 45% acetic acid at 60°C for 30 s, stained with 1% aceto-orcein overnight, and squashed for chromosome observations. The preparations obtained were examined and photographed using a Leica DM1000 microscope (Leica Microsystems, Wetzlar, Germany).

Metaphasic plates or idiograms were analyzed with KaryoType software (http://mnh.scu.edu.cn/soft/blog/KaryoType/index.html) and lengths of individual chromosomes were measured. Using karyotype data for each species, chromosome pairs were arranged in order of decreasing length, and then the absolute lengths of the long and short arms of each chromosome pair were measured. The data were then pooled according to the considered taxonomic groups. In order to provide the general phenotypic appearance of karyotypes, a mean haploid idiogram (see also Peruzzi et al. 2009) was built for each sample.

The designation of the centromeric position as median (m), submedian (sm), or subterminal (st) followed methods described by Levan et al. (2010), and the karyotype asymmetries were estimated according to Stebbins (1971) and Paszko (2006). The following parameters were calculated for the numerical characteristics of the karyotypes: (1) shortest (SC) and longest (LC) chromosome length; (2) ratio of the longest to shortest chromosome (LC/SC); (3) mean long arm length (p); (4) mean short arm length (q); (5) mean total chromosome length (CL); (6) asymmetry index (AI) = CVCL × CVCI/100 (Paszko 2006); (7) Stebbins’ type (KA); and (8) karyotypic formula (Table 1).

3. Results

Detailed chromosome parameters of studied taxa are shown in Table 1, and karyomorphology and idiograms are illustrated in Figures 1–48.

Table 1. Sample information and karyotypic characteristics of 18 Epimedium species examined in this study.

Species	Vouchers	Origin	SC-LC	LC/SC	p	q	CL	KA	AI	Karyotype formula	Figures
E. acuminatum Franchet	Zhang 232	Emei, Sichuan	2.93–4.36	1.49	2.32 ± 0.30	1.34 ± 0.34	3.67 ± 0.39	3A	2.26	6m (2sat) + 6sm	1, 25
E. baojingense Q. L. Chen & B. M. Yang	Zhang 203	Longshan, Hunan	4.06–5.38	1.33	2.89 ± 0.21	1.77 ± 0.49	4.67 ± 0.46	2A	2.14	6m (2sat) + 6sm	2, 26
E. brevicornu Maximowicz	Zhang 201	Shangnan, Shaanxi	4.03–5.84	1.45	3.19 ± 0.35	1.76 ± 0.49	4.94 ± 0.52	3A	2.37	4m (2sat) + 8sm	3, 27
E. franchetii Stearn	Zhang 166	Wufeng, Hubei	3.9–5.95	1.52	2.74 ± 0.20	1.83 ± 0.52	4.57 ± 0.64	2A	2.68	6m (2sat) + 6sm	4, 28
E. franchetii Stearn	Zhang 173	Wufeng, Hubei	3.26–4.61	1.42	2.47 ± 0.14	1.51 ± 0.41	3.98 ± 0.48	2A	2.28	6m (2sat) + 6sm	5, 29
E. franchetii Stearn	Zhang 196	Shennongjia, Hubei	3.28–4.77	1.45	2.45 ± 0.21	1.60 ± 0.46	4.04 ± 0.48	2A	2.57	6m (2sat) + 6sm	6, 30
E. hunanense (Handel Mazzetti) Handel-Mazzetti	Zhang 207	Yongshun, Hunan	3.73–5.87	1.57	2.87 ± 0.26	1.70 ± 0.64	4.6 ± 0.77	2A	4.56	6m (2sat) + 4sm + 2st	7, 31
E. ilicifolium Stearn	Zhang 200	Zhuxi, Hubei	3.24–4.95	1.53	2.60 ± 0.33	1.59 ± 0.45	4.80 ± 0.54	3A	2.88	6m (2sat) + 6sm	8, 32
E. leptorrhizum Stearn	Zhang 183	Longshan, Hunan	2.88–4.04	1.4	2.01 ± 0.12	1.35 ± 0.38	3.37 ± 0.34	2A	2.19	6m (2sat) + 6sm	9, 33
E. leptorrhizum Stearn	Zhang 152	Longquan, Zhejiang	2.87–4.57	1.59	2.31 ± 0.20	1.47 ± 0.48	3.78 ± 0.51	3A	3.31	6m (2sat) + 6sm	10, 34
E. leptorrhizum Stearn	Zhang 211	Enshi, Hubei	4–5.76	1.44	3.10 ± 0.43	1.77 ± 0.40	4.88 ± 0.48	2A	1.94	4m + 8sm (2sat)	11, 35
E. lishihchenii Stearn	Zhang 149	Lushan, Jiangxi	3.99–4.5	1.51	2.23 ± 0.19	1.37 ± 0.35	3.59 ± 0.47	2A	2.1	6m(2sat) + 6sm	12, 36
E. membranaceum K. Meyer	Zhang 275	Dujiangyan, Sichuan	3.21–4.54	1.41	2.39 ± 0.29	1.48 ± 0.35	3.88 ± 0.42	2A	2.23	6m (2sat) + 6sm	13, 37
E. mikinorii Stearn	Zhang 187	Enshi, Hubei	3.45–5.02	1.45	2.72 ± 0.30	1.60 ± 0.44	4.32 ± 0.50	2A	2.4	6m (2sat) + 6sm	14, 38
E. parvifolium S. Z. He &T. L. Zhang	Zhang 218	Songtao, Guizhou	3.13–5.53	1.77	2.46 ± 0.34	1.59 ± 0.44	4.05 ± 0.65	2A	2.76	6m (2sat) + 6sm	15, 39
E. pseudowushanense B. L. Guo	Zhang 228	Longli, Guizhou	3.81–5.68	1.49	2.80 ± 0.25	1.81 ± 0.43	4.61 ± 0.49	2A	1.71	6m (2sat) + 6sm	16, 40
E. sagittatum (Siebold & Zuccarini) Maximowicz	Zhang 215	Youyang, Chongqing	3.78–5.65	1.49	2.76 ± 0.20	1.77 ± 0.53	4.54 ± 0.57	2A	2.61	6m (2sat) + 6sm	17, 41
E. shennongjiaense Y. J. Zhang & J. Q. Li	Zhang 193	Shennongjia, Hubei	3.72–5.14	1.38	2.68 ± 0.40	1.56 ± 0.30	4.23 ± 0.41	2A	1.85	4m + 8sm (2sat)	18, 42
E. stellulatum Stearn	Zhang 198	Shiyan, Hubei	3.6–5.56	1.54	2.76 ± 0.36	1.70 ± 0.37	4.45 ± 0.54	2A	1.9	8m (2sat) + 4sm	19, 43
E. stellulatum Stearn	Zhang 202	Luonan, Shannxi	3.45–4.99	1.45	2.61 ± 0.18	1.58 ± 0.49	4.19 ± 0.58	2A	2.89	6m (2sat) + 6sm	20, 44
E. tianmenshanensis T. Deng, D. G. Zhang & H. Sun	Zhang 209	Zhangjiajie, Hunan	3.47–5.42	1.56	2.67 ± 0.25	1.72 ± 0.58	4.38 ± 0.61	2A	3.71	6m (2sat) + 6sm	21, 45
E. wushanense T. S. Ying	Zhang 191	Shennongjia, Hubei	3.83–5.64	1.47	2.76 ± 0.39	1.76 ± 0.40	4.52 ± 0.54	2A	1.9	8m (2sat) + 4sm	22, 46
E. wushanense T. S. Ying	Zhang 192	Shennongjia, Hubei	2.97–4.25	1.43	2.33 ± 0.33	1.31 ± 0.30	3.64 ± 0.41	2A	2.22	4m + 8sm (2sat)	23, 47
E. zhushanense K. F. Wu & S. X. Qian	Zhang 199	Zhushan, Hubei	3.66–5.78	1.58	3.07 ± 0.41	1.78 ± 0.50	4.85 ± 0.65	2A	2.76	8m (2sat) + 4sm	24, 48

Abbreviations: SC, the shortest chromosome length (μm); LC, the longest chromosome length (μm); LC/SC, proportion of the longest chromosome length to the shortest chromosome length; p, mean length of long arm (μm); q, mean length of short arm (μm); CL, mean length of chromosome (μm); AI, asymmetry induce according to Paszko (2006); KA, Stebbins’ asymmetry karyotype; m, metacentric; sm, submetacentric; st, acrocentric.

3.1. Epimedium acuminatum Franchet

The karyotype formula of E. acuminatum collected from the Emei Mountains in Sichuan is 2n = 2x = 12 = 6m (2sat) + 6sm. The first, third, and fifth chromosome pairs are metacentric and the others are submetacentric as observed at mitotic metaphase. Intercalary satellites were observed on the first pair of chromosomes. The total haploid length of the chromosomes is 22.00 μm with single chromosome lengths ranging from 2.93 to 4.36 μm. The Stebbins’ asymmetry is categorized as 3A and the AI = 2.26 (Table 1). The mitotic metaphase chromosomes and idiogram are shown in Figures 1 and 25, respectively.

Figures 1-24. Metaphase chromosomes of the 18 examined species in Epimedium. 1, E. acuminatum; 2, E. baojingense; 3, E. brevicornu; 4–6, E. franchetii; 7, E. hunanense; 8, E. ilicifolium; 9–11, E. leptorrhizum; 12, E. lishihchenii; 13, E. membranaceum; 14, E. mikinorii; 15, E. parvifolium; 16, E. pseudowushanense; 17, E. sagittatum; 18, E. shennongjiaense; 19, 20, E. stellulatum; 21, E. tianmenshanensis; 22, 23, E. wushanense; 24, E. zhushanense. Scale bar = 5 μm.

Figure 25-48. Idiograms of the 18 species of Epimedium examined in this study. 25, E. acuminatum; 26, E. baojingense; 27, E. brevicornu; 28–30, E. franchetii; 31, E. hunanense; 32, E. ilicifolium; 33–35, E. leptorrhizum; 36, E. lishihchenii; 37, E. membranaceum; 38, E. mikinorii; 39, E. parvifolium; 40, E. pseudowushanense; 41, E. sagittatum; 42, E. shennongjiaense; 43, 44, E. stellulatum; 45, E. tianmenshanensis; 46, 47, E. wushanense; 48, E. zhushanense. Scale bar = 2 μm.

3.2. Epimedium baojingense Q. L. Chen & B. M. Yang

The somatic chromosome number of E. baojingense is 2n = 2x = 12 = 6m (2sat) + 6sm, with satellites observed in the interstitial region of the first pair of chromosomes. The first, second, and fifth chromosome pairs are metacentric and the others are submetacentric as observed at mitotic metaphase. The individual chromosome lengths range between 4.06 and 5.38 μm, with a total haploid length of 27.98 μm. The Stebbins’ asymmetry category is 2A and the AI = 2.14 (Table 1). The mitotic metaphase chromosomes and idiogram are shown in Figures 2 and 26, respectively.

3.3. Epimedium brevicornu Maximowicz

Epimedium brevicornu collected from Shanxi has a somatic karyotype formula of 2n = 2x = 12 = 4m (2sat) + 8sm. Its first and third chromosome pairs are metacentric and the others are submetacentric as observed at mitotic metaphase. Satellites were observed in the interstitial region of the second pair of chromosomes. The chromosome lengths range between 4.03 and 5.84 μm and the total haploid length of the chromosome is 29.65 μm. The Stebbins’ asymmetry is categorized as 3A and the AI = 2.37 (Table 1). The mitotic metaphase chromosomes and idiogram are given in Figures 3 and 27, respectively.

3.4. Epimedium franchetii Stearn

Three samples of E. franchetii were included in this study, and two types of karyotype formulas and different AI values were observed; the same Stebbins’ asymmetry category was found (Table 1). The first sample, collected from Wufeng, Hubei, has a somatic chromosome number of 2n = 2x = 12 = 6m (2sat) + 6sm. In this sample, the first, second, and third chromosome pairs are metacentric and the others are submetacentric as observed at mitotic metaphase. Intercalary satellites were observed on the first pair of chromosomes. The chromosomes lengths range between 3.9 and 5.95 μm and the total haploid length of the chromosome is 27.41 μm. The Stebbins’ asymmetry category is 2A and the AI = 2.68 (Table 1). The mitotic metaphase chromosomes and idiogram are given in Figures 4 and 28, respectively.

The second sample, also collected from Wufeng, Hubei, has a somatic karyotype formula of 2n = 2x = 12 = 6m (2sat) + 6sm. At mitotic metaphase, the first, second and third chromosome pairs are metacentric and three pairs of submetacentric chromosomes were observed. Intercalary satellites were observed on the second pair of chromosomes. The chromosome lengths range between 3.26 and 4.61 μm. The total haploid length of the chromosome is 23.86 μm. The Stebbins’ asymmetry is 2A and the AI = 2.28 (Table 1). The mitotic metaphase chromosomes and idiogram are shown in Figures 5 and 29, respectively.

The third sample was collected from Shennongjia, Hubei. The somatic karyotype formula is 2n = 2x = 12 = 6m (2sat) + 6sm and its first, second, and third chromosome pairs are metacentric and the others are submetacentric as observed in mitotic metaphase. Satellites were observed in the interstitial region of the first pair of chromosomes. The chromosome lengths range between 3.28 and 4.77 μm and the total haploid length of the chromosome is 24.26 μm. The Stebbins’ asymmetry is classified to 2A and the AI = 2.57 (Table 1). The mitotic metaphase chromosomes and idiogram are given in Figures 6 and 30, respectively.

3.5. Epimedium hunanense (Handel-Mazzetti) Handel-Mazzetti

The chromosome number and karyotype is first reported for E. hunanense. The mitotic metaphase chromosomes and idiogram for this species are given in Figures 7 and 31, respectively. Its somatic karyotype formula is 2n = 2x = 12 = 6m (2sat) + 4sm + 2st. At mitotic metaphase, the first, the second and third chromosome pairs are metacentric, the fourth, and fifth pairs are submetacentric, and the sixth pair is subteleocentric. Satellites were observed in the interstitial region of the first pair of chromosomes. Chromosome lengths range between 3.73 and 5.87 μm. The total haploid length of the chromosome is 27.44 μm. The Stebbins’ asymmetry is categorized as 2A and the AI = 4.56 (Table 1).

3.6. Epimedium ilicifolium Stearn

The somatic karyotype formula of E. ilicifolium is 2n = 2x = 12 = 6m (2sat) + 6sm. At mitotic metaphase, the first, third and fourth chromosome pairs are metacentric, the second, fifth, and sixth pairs are submetacentric. Satellites were found in the interstitial region of the first pair of chromosomes. The chromosome lengths range between 3.24 and 4.95 μm and the total haploid length of the chromosome is 25.08 μm. The Stebbins’ asymmetry category is 3A and the AI = 2.88 (Table 1). The mitotic metaphase chromosomes and idiogram are presented in Figures 8 and 32, respectively.

3.7. Epimedium leptorrhizum Stearn

Three samples of E. leptorrhizum were investigated, showing two types of karyotype formulas, different AI values, and different Stebbins’ asymmetry categories. The first sample, collected from Longshan, Hunan, has a chromosome number of 2n = 2x = 12 = 6m (2sat) + 6sm. At mitotic metaphase, the first, second, and third chromosome pairs are metacentric and the others are submetacentric. Satellites were observed in the interstitial region of the second pair of chromosomes. The chromosome lengths range between 2.88 and 4.04 μm with a total haploid length of 29.25 μm. The Stebbins’ asymmetry category is classified as 2A and the AI = 2.19 (Table 1). The mitotic metaphase chromosomes and idiogram are given in Figures 9 and 33, respectively.

Epimedium leptorrhizum was only recently reported in Zhejiang (Xia et al. 2011). The sample from Longquan, Zhejiang has a somatic karyotype formula of 2n = 2x = 12 = 6m (2sat) + 6sm. At mitotic metaphase, the first, second, and third chromosome pairs are metacentric and the others are submetacentric. Satellites were observed in the interstitial region of the first pair of chromosomes. The chromosome lengths range between 2.87 and 4.57 μm with a total haploid length of 20.19 μm. The Stebbins’ asymmetry category is 3A and the AI = 3.31 (Table 1). The mitotic metaphase chromosomes and idiogram are given in Figures 10 and 34, respectively.

The third sample, collected from Enshi, Hubei, has a somatic karyotype formula of 2n = 2x = 12 = 4m + 8sm (2sat) with two pairs of metacentric and four pairs of submetacentric chromosomes. Satellites were observed in the interstitial region of the first pair of chromosomes. The chromosome lengths range between 4 and 5.76 μm and the total haploid length is 22.69 μm. The Stebbins’ asymmetry is 2A and the AI = 1.94 (Table 1). The mitotic metaphase chromosomes and idiogram are shown in Figures 11 and 35, respectively.

3.8. Epimedium lishihchenii Stearn

The mitotic metaphase chromosomes and idiogram of E. lishihchenii are given in Figures 12 and 36, respectively. Its somatic karyotype formula is 2n = 2x = 12 = 6m (2sat) + 6sm. The first, second, and fourth chromosome pairs are metacentric and the others are submetacentric as observed at mitotic metaphase. The second pair of chromosomes bear satellites at an intercalary position. The chromosome lengths range from 2.98 to 4.5 μm and the total haploid length of the chromosome is 21.56 μm. The Stebbins’ asymmetry is 2A and the AI = 2.10 (Table 1). The mitotic metaphase chromosomes and idiogram are given in Figures 12 and 36, respectively.

3.9. Epimedium membranaceum K. Meyer

The chromosome number and karyotype of E. membranaceum was counted for the first time with samples collected from Dujiangyan, Sichuan. The karyotype formula is 2n = 2x = 12 = 6m (2sat) + 6sm. The first, second, and third chromosome pairs are metacentric and the others are submetacentric as observed at mitotic metaphase. Satellites are located in the interstitial region of the first pair of chromosomes. The chromosome lengths range between 3.21 and 4.54 μm and the total haploid length of the chromosome is 23.25 μm. The Stebbins’ asymmetry is categorized as 2A and the AI = 2.23 (Table 1). The mitotic metaphase chromosomes and idiogram are presented in Figures 13 and 37, respectively.

3.10. Epimedium mikinorii Stearn

Epimedium mikinorii, collected from Enshi, Hubei, has a somatic karyotype formula of 2n = 2x = 12 = 6m (2sat) + 6sm. At mitotic metaphase, the first, second, and third chromosome pairs are metacentric and the others are submetacentric. Satellites are located in the interstitial region of the first pair of chromosomes. The chromosome lengths range between 3.45 and 5.02 μm, with a total haploid length of 25.91 μm. The Stebbins’ asymmetry category is 2A and the AI = 2.40 (Table 1). The mitotic metaphase chromosomes and idiogram are shown in Figures 14 and 38, respectively.

3.11. Epimedium parvifolium S. Z. He & T. L. Zhang

The somatic karyotype formula of E. parvifolium is 2n = 2x = 12 = 6m (2sat) + 6sm. The first, second, and fourth chromosome pairs are metacentric and the others are submetacentric as observed at mitotic metaphase. Satellites were observed in the interstitial region of the first pair of chromosomes. The chromosome lengths range between 3.13 and 5.53 μm. The total haploid length of the chromosome is 24.29 μm. The Stebbins’ asymmetry is 2A and the AI = 2.76 (Table 1). The mitotic metaphase chromosomes and idiogram are shown in Figures 15 and 39, respectively.

3.12. Epimedium pseudowushanense B. L. Guo

The chromosome number and karyotype formula of E. pseudowushanense from Longli, Guizhou is 2n = 2x = 12 = 6m (2sat) + 6sm. At mitotic metaphase, the first, second, and third chromosome pairs are metacentric and the others are submetacentric. Satellites were observed in the interstitial region of the first pair of chromosomes. The chromosome lengths range between 3.81 and 5.68 μm. The total haploid length of the chromosome is 27.63 μm. The Stebbins’ asymmetry is categorized as 2A and the AI = 1.71 (Table 1). The mitotic metaphase chromosomes and idiogram are given in Figures 16 and 40, respectively.

3.13. Epimedium sagittatum (Siebold & Zuccarini) Maximowicz

The somatic karyotype formula of this sample from Youyang, Chongqing is reported with 2n = 2x = 12 = 6m (2sat) + 6sm. The first, second, and third chromosome pairs are metacentric and the others are submetacentric as observed at mitotic metaphase. Satellites are located in the interstitial region of the first pair of chromosomes. The chromosome lengths range from 3.78 to 5.65 μm with the total haploid length of 27.23 μm. The Stebbins’ asymmetry is 2A and the AI = 2.66 (Table 1). The mitotic metaphase chromosomes and idiogram are presented in Figures 17 and 41, respectively.

3.14. Epimedium shennongjiaense Y. J. Zhang & J. Q. Li

Epimedium shennongjiaense, a new species reported by Zhang and Li (2009), is endemic to Shennongjia, Hubei Province. The chromosome number is counted here for the first time with the somatic karyotype formula of 2n = 2x = 12 = 4m + 8sm (2sat). The second and third pairs of chromosomes are metacentric and the others are submetacentric as observed at mitotic metaphase. Satellites were found in the interstitial region of the first pair of chromosomes. The chromosome lengths range from 3.72 to 5.14 μm and the total haploid length is measured as 25.40 μm. The Stebbins’ asymmetry category is 2A and the AI = 1.85 (Table 1). The mitotic metaphase chromosomes and idiogram are shown in Figures 18 and 42, respectively.

3.15. Epimedium stellulatum Stearn

Two samples of E. stellulatum were included here, having different karyotype formulas and AI values, but the same Stebbins’ asymmetry. The somatic karyotype formula of the sample from Wudang Mountain, Hubei is 2n = 2x = 12 = 8m (2sat) + 4sm. It has four pairs of metacentric and two pairs of submetacentric chromosomes found at mitotic metaphase. Satellites were observed in the interstitial region of the first pair of chromosomes. Its chromosome lengths range between 3.6 and 5.56 μm with the total haploid length of 26.72 μm. The Stebbins’ asymmetry is 2A and the AI = 1.90 (Table 1). The mitotic metaphase chromosomes and idiogram are shown in Figures 19 and 43, respectively.

The second sample was collected from Luonan, which is a new record in Shaanxi. The somatic karyotype formula is 2n = 2x = 12 = 6m (2sat) + 6sm. Three pairs of metacentric (1–3) chromosomes were observed and the other chromosomes were submetacentric as investigated at mitotic metaphase. Satellites were located in the interstitial region of the first pair of chromosomes. The chromosome lengths range between 3.45 and 4.99 μm and the total haploid length is 25.15 μm. The Stebbins’ asymmetry is categorized as 2A and the AI = 2.89 (Table 1). The mitotic metaphase chromosomes and idiogram are given in Figures 20 and 44, respectively.

3.16. Epimedium tianmenshanensis T. Deng, D. G. Zhang & H. Sun

Epimedium tianmenshanensis is a recently described species (Zhang et al. 2015), endemic to the Tianmen Mountain in western Hunan Province. Its somatic karyotype formula was first examined here: 2n = 2x = 12 = 6m (2sat) + 6sm. The first, second, and third pairs of chromosomes are metacentric and the others are submetacentric at mitotic metaphase. Satellites were observed in the interstitial region of the first pair of chromosomes. The chromosome lengths range between 3.47 and 5.42 μm and the total haploid length of the chromosome is 26.29 μm. The Stebbins’ asymmetry is 2A category and the AI = 3.71 (Table 1). The mitotic metaphase chromosomes and idiogram are presented in Figures 21 and 45, respectively.

3.17. Epimedium wushanense T. S. Ying

Two counts were reported for this species with different karyotype formulas and AI values, but the same Stebbins’ asymmetry. The first sample was collected from a low elevation (500 m) area of Xiagu Town, Shennongjia National Nature Reserve, Hubei. The somatic karyotype formula is 2n = 2x = 12 = 8m (2sat) + 4sm. At mitotic metaphase, the first, second, fourth, and fifth pairs of chromosomes are metacentric and the others are submetacentric. Satellites were observed in the interstitial region of the first pair of chromosomes. The chromosome lengths range from 3.83 to 5.64 μm. The total haploid length of the chromosome is 27.12 μm. The asymmetry category is Stebbins’ 2A and the AI = 1.90 (Table 1). The mitotic metaphase chromosomes and idiogram are shown in Figures 22 and 46, respectively.

The second sample was collected from Shennongjia National Nature Reserve in Hubei. Its somatic karyotype formula is 2n = 2x = 12 = 4m + 8sm (2sat). The second and third chromosome pairs are metacentric and the other chromosomes are submetacentric as found at mitotic metaphase. Satellites were observed in the interstitial region of the first pair of chromosomes. The chromosome lengths range from 2.97 to 4.25 μm and the total haploid length is 21.80 μm. The Stebbins’ asymmetry is 2A and the AI = 2.22 (Table 1). The mitotic metaphase chromosomes and idiogram are shown in Figures 23 and 47, respectively.

3.18. Epimedium zhushanense K. F. Wu & S. X. Qian

Epimedium zhushanense is endemic to Hubei and its somatic chromosome number and karyotype formula is reported as 2n = 2x = 12 = 4m + 8sm (2sat). The second and third pairs of chromosomes are metacentric and the others are submetacentric as observed at mitotic metaphase. Satellites are located in the interstitial region of the first pair of chromosomes. The chromosome lengths range between 3.66 and 5.78 μm and its total haploid length is 29.09 μm. The Stebbins’ asymmetry is categorized as 2A and the AI = 2.76 (Table 1). The mitotic metaphase chromosomes and idiogram are given in Figures 24 and 48, respectively.

4. Discussion

This study reported chromosome numbers and karyotypes of 18 species of Epimedium from 24 populations (Table 1; Figure 1–48), all from sect. Diphyllon and all endemic to China. The cytological data confirmed that the chromosome numbers of E. acuminatum, E. baojingense, E. brevicornu, E. franchetii, E. ilicifolium, E. leptorrhizum, E. mikinorii, E. parvifolium, E. pseudowushanense, E. sagittatum, E. stellulatum, E. wushanense, and E. zhushanense are diploid (2n = 12) with the basic number of x = 6. Chromosome numbers of five species were reported for the first time (i.e. E. hunanense, E. lishihchenii, E. membranaceum, E. shennongjiaense, and E. tianmenshanensis) with 2n = 2x = 12 (Table 1).

Sheng et al. (2010) reported the karyotype of Epimedium lishihchenii sampled from Tongren, Guizhou, with the somatic karyotype formula of 2n = 2x = 12 = 4m (2sat) + 8sm. However, their sample might be misidentified because E. lishihchenii is endemic to Jiangxi. Our material was collected from Lushan, Jiangxi with the chromosome number and karyotype correctly reported for the first time.

Combined with results reported in earlier studies (e.g. Tanaka and Takahashi 1981; Takahashi 1989; Sheng and Chen 2007, 2008; Sheng et al. 2010; Yan et al. 2016), cytological data has suggested that Epimedium has an evolutionary history of diploidy with 2n = 2x = 12, except for E. yingjiangense with 2n = 4x = 24 (Sheng and Chen 2007; Sheng and Tian 2011), but E. yingjiangense is also known to have 2n = 2x = 12 (Sheng and Chen 2007).

All Epimedium species examined in this study present a similar karyomorphology including only metacentric and submetacentric chromosomes, except for E. hunanense with its sixth chromosome pair subteleocentric with an arm ratio of 3.17 (Table 1). Previous studies also confirm similar karyotypes for Epimedium (Tanaka and Takahashi 1981; Takahashi 1989; Sheng and Chen 2007, 2008; Zhang et al. 2008; Sheng et al. 2010). In the present study, intercalary satellites were commonly found in the first pair in all species, except for E. leptorrhizum, E. lishihchenii, E. brevicornu, and E. franchetii, which had a secondary constriction in the second pair. The length of pairs 1 and 2 for the above-mentioned four species was similar. Combined with previous karyotype investigations on Epimedium (Tanaka and Takahashi 1981; Takahashi 1989; Sheng and Chen 2007, 2008; Zhang et al. 2008; Diao et al. 2011), we suggest that chromosome pair 2 of these four species was congenetic with pair 1 of the other species, which is similar to results found in Zhang et al. (2008) and Sheng et al. (2010).

Karyotype asymmetry indices have been widely used to make assumptions about the mechanisms of chromosomal evolution in plants (Paszko 2006). The Stebbins’ (1971) classification is the most frequently used qualitative method for assessing symmetry conditions and describing the karyotypic relationships between different taxa. In this study, all of the investigated species display relatively primitive 2A asymmetry, except E. acuminatum, E. brevicornu, E. ilicifolium, and one accession of E. leptorrhizum, which showed 3A asymmetry (Table 1). Other previous studies have found a few species (E. baojingense, E. glandulosopilosum, E. leptorrhizum, E. pauciflorum, E. shuichengense, and E. yingjiangense with 1A asymmetry; E. dolichostemon with 3A asymmetry) with varying Stebbins’ classifications (Tanaka and Takahashi 1981; Takahashi 1989; Sheng and Chen 2007, 2008). As a result, the karyotypes of Epimedium sect. Diphyllon species investigated was highly uniform and primitive, as suggested by previous reports on Epimedium (Tanaka and Takahashi 1981; Takahashi 1989; Sheng and Chen 2007, 2008). Due to the uniform karyotype and the limited Stebbins (1971) qualitative classification, it is difficult to infer the phylogenetic relationship within Epimedium solely based on karyotypic evidence.

Previous studies have suggested stable cytological results for species such as E. acuminatum, E. ecalcaratum, E. pubescens, and E. sagittatum (Sheng and Chen 2007, 2008; Zhang et al. 2008; Sheng et al. 2010; Diao et al. 2011). To further examine the chromosome variation within Epimedium species, more than two individuals were sampled for E. franchetii, E. leptorrhizum, E. stellulatum, and E. wushanense (Table 1). These species had consistent and stable cytological traits with respect to their karyotype formula, Stebbins’ asymmetry, and AI values, except for E. leptorrhizum. The two accessions of E. leptorrhizum from Hunan and Hubei have similar karyotypes and 2A asymmetry, but the third accession from Zhejiang had 3A type asymmetry and the AI = 3.31 (Table 1). The sample collected from Zhejiang is morphologically similar to the samples from Hunan and Hubei, but had relatively large flowers. Thus, further molecular phylogenetic studies are merited to test the status of the population of E. leptorrhizum from Zhejiang. Furthermore, this species has previously been reported with Stebbins 1A asymmetry (Sheng et al. 2010), which might indicate that chromosome variation is large within this species.

A recent molecular phylogeny of Epimedium conducted by Zhang et al. (2014b) suggested that the two subgenera and four sections are monophyletic and Epimedium sect. Diphyllon was divided into five well-supported clades. This study also showed that five species (E. brevicornu, E. elongatum, E. pubescens, E. stellulatum, and E. sutchuenense) are either isolated or formed a polytomy located near the root of the section. Due to the uniform karyotypes and similar asymmetry in sect. Diphyllon, we found no distinct correlation between the karyotypic data and phylogenetic relationships among these Chinese species of Epimedium. Furthermore, we used a newer asymmetry index developed by Paszko (2006), which has advantages of allowing a high degree of precision and sensitivity when assessing karyotype asymmetry. The AI values of the taxa included in this study range from 1.71 to 4.56, but no association was found with morphological characters or biogeographic distribution. As a result, it is difficult to discuss the phylogenetic evolution within Chinese Epimedium species based on the cytological results. Further evidence, such as fluorescent banding and physical mapping of 45S and 5S rDNA as shown in Sheng and Wang (2010) and Diao et al. (2011), provide promising advances in elucidating the evolutionary relationships among Epimedium species on the chromosomal structural level.

Acknowledgments

We thank Min Gao, Li-Ying Luo, and Zhen-Xin Xie for assistance in cytological lab works and Meng-Hua Zhang for her valuable comments on the early version of this manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was supported by grants from the Nature Sciences Foundation of China [31560060, 31570211], the educational reform project of Jishou University [2016SYJG014, 2016KCB13, JG2017B01], the National Special Fund on Basic Research of Science and Technology of China [2014FY110100], Traditional Chinese Medicine Public Health Special Project [[2011]76, 201207002], Heavy Metal Pollution Control and Ecological Restoration in Xiangxi Mining for Graduate Training Innovation Base of Hunan Province, and Jishou University Undergraduate Research Innovation Project [JDX17023].