Taxonomic and phylogenetic analysis of Epimedium L. based on amplified fragment length polymorphisms

Scientia Horticulturae 170 (2014) 284–292

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Taxonomic and phylogenetic analysis of Epimedium L. based on amplified fragment length polymorphisms Yanjun Zhang a , Lulu Yang a , Jianjun Chen a , Wei Sun b , Ying Wang a,∗ a Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China b Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Science, Beijing 100700, China

a r t i c l e

i n f o

Article history: Received 16 August 2013 Received in revised form 18 February 2014 Accepted 24 February 2014 Available online 13 April 2014 Keywords: Epimedium AFLP Taxonomy Phylogeny

a b s t r a c t Epimedium is well known for its ornamental and medicinal value. The genus consists of ca. 58 species disjunctly distributed in the Mediterranean region and eastern Asia, with the highest species diversity concentrated in central-southeastern China. In the present research, we collected 144 accessions from 58 Epimedium species and one accession representing the outgroup Vancouveria hexandra. Using Bayesian analysis, the phylogeny of Epimedium was reconstructed based on amplified fragment length polymorphism data. The dendrogram suggested that two subgenera and four sections of Epimedium were monophyletic. Chinese sect. Diphyllon was divided into five well-supported clades related to flower morphology except that five species were either isolated or formed a general polytomy. The result also well supported the recent morphological revision on E. reticulatum, E. wushanense, E. ilicifolium, E. jinchengshanense, E. simplicifolium, E. chlorandrum, E. brachyrrhizum, and E. dewuense, and provided significant implications for E. sagittatum complex. The present research is of great implication for facilitating the utilization of natural germplasm of Epimedium, especially for further development of new cultivars for ornamental and medicinal purposes. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Epimedium species has been known as an exotic perennial garden plant in western countries and also used as a herbal medicine in Asian countries. Bearing attractive foliage and flowers, Epimedium has become a popular shading plant with great commercial prospects (Lubell and Brand, 2005; Ren et al., 2008; Avent, 2010). Meanwhile, modern pharmaceutical studies have verified its wide-reaching activity against sexual dysfunction, osteoporosis, cardiovascular diseases, menstrual irregularity, asthma, chronic nephritis, and immunoregulation (Ma et al., 2011; Gao et al., 2012; Li et al., 2012; Yin et al., 2012). The recent focus on the research and development of Epimedium has prompted the need for in-depth taxonomic and phylogenetic studies of the genus, especially for the highly diversified Chinese taxa (Shen et al., 2007; Guo et al., 2008; Govindaraghavan et al., 2012). The genus Epimedium L. (Berberidaceae) contains approximately 58 species of herbs in the North Temperate Zone, distributing disjunctly and very unevenly in the Mediterranean

∗ Corresponding author. Tel.: +86 27 87510675; fax: +86 27 87510670. E-mail address: [email protected] (Y. Wang). http://dx.doi.org/10.1016/j.scienta.2014.02.025 0304-4238/© 2014 Elsevier B.V. All rights reserved.

region and eastern Asia (Stearn, 2002; Ying et al., 2011). Based on flower and leaf morphology, C-banding of chromosomes (Takahashi, 1989), and geographical distribution, the updated system of the genus comprised two subgenera, four sections, and four series (Stearn, 2002). Subgenus Rhizophyllum comprised E. perralderianum endemic to Algeria and E. pinnatum from Caucasia. Subgenus Epimedium consisted of four sections: (1) section Epimedium with E. alpinum in Alps and Balkan areas and E. pubigerum from Caucasia; (2) section Polyphyllon, comprising E. elatum, limited to the western Himalaya; (3) section Macroceras with six species distributed in Japan, Korea, northeastern China, and Far Eastern Russia; and (4) section Diphyllon, with about 47 known species in central-southeastern China, was further subdivided into four series based mainly on flower morphology, particularly on the petal characteristics (Fig. 1). China is the diversity center of Epimedium, containing over 80% of species of the genus (Stearn, 2002; Ying, 2002; Ying et al., 2011). Chinese Epimedium has presented a number of taxonomic questions with about 30 species published in the past 30 years (Ying et al., 2011; Govindaraghavan et al., 2012). The poor quality of type specimen and inadequate investigation for some species might result in inaccurate morphological descriptions, controversial subgenera classification, and publication of synonymous new

Y. Zhang et al. / Scientia Horticulturae 170 (2014) 284–292

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Fig. 1. Floral photos of 11 species from four series of Chinese Epimedium of sect. Diphyllon. (A) E. platypetalum, (B) E. ecalcaratum, (C) E. fangii, (D) E. mikinorii, (E) E. franchetii, (F)-(G) E. acuminatum, (H) E. fargesii, (I) E. dolichostemon, (J) E. brevicornu, (K) E. pubescens, (L) E. sagittatum. (A) and (B) belonging to ser. Campanulatae, (C) and (D) belonging to ser. Davidianae, (E)-(G) belonging to ser. Dolichocerae, (H)-(L) belonging to ser. Brachycerae. Scale bar = 5 mm.

species (Guo et al., 2008; Ying et al., 2011; Zhang et al., 2011; Smet et al., 2012). Furthermore, Stearn, 2002 classified Chinese Epimedium species (sect. Diphyllon) into four series according to flower morphology. However, phylogenetic studies could not confirm the classification for Chinese species based on pollen types, flavonoid types, karyotypes, and molecular markers (Guo et al., 1998, 2008; Guo and Xiao, 1999; Sun et al., 2005; Shen et al., 2007; Zhang et al., 2007, 2008; Sheng et al., 2010, 2011b; Smet et al., 2012). In addition, Stearn, 2002 classified Epimedium into two subgenera and four sections, but molecular phylogenetic analyses consistently suggested that subgen. Rhizophyllum was nested within subgen. Epimedium. Although the four sections were supported, their relationships were either unresolved or poorly supported but sect. Epimedium as sister to sect. Macroceras (Sun et al., 2005; Zhang et al., 2007; Smet et al., 2012). Amplified fragment length polymorphism (AFLP) is a wholegenome approach for studying genetic variation that can provide much better resolution than nuclear ribosomal and chloroplast DNA sequence data at lower-level systematics (De Cock et al., 2008; Koopman et al., 2008; Mckinnon et al., 2008; Bog et al., 2010). In previous researches, AFLP was used to elucidate the phylogeny of Epimedium, but resulted in an unresolved polytomy for the Chinese taxa due to a very limited number of markers (Shen et al., 2007; Smet et al., 2012). In the last 10 years, an intensive study on the morphological taxonomy of Chinese Epimedium species has been conducted, but no molecular markers were employed to verify these morphology-based revisions (Zhang et al., 2011; Zhang et al.,

unpublished data). In the present study, more than 500 polymorphic AFLP fragments, yielded by eight primer combinations, were analyzed in the most complete sampling of Epimedium species to date. We aimed to address these questions: (1) Can AFLP analysis support the subscription of two subgenera and four sections of Epimedium? (2) Can AFLP analysis resolve the subscription of four series of Chinese Epimedium? (3) Does AFLP analysis support the morphological taxonomy of Chinese Epimedium? 2. Materials and methods 2.1. Plant materials We used 144 accessions from 58 Epimedium species that covered all the species of the genus, and used one accession from Vancouveria hexandra as outgroup. Detailed sample information is provided in Table 1, and arranged according to the classification of Stearn, 2002. The grouping of E. reticulatum, E. shuichengense, E. wushanense, and E. ilicifolium was adjusted based on our recent morphological revision (Zhang et al., unpublished data). 2.2. DNA extraction and AFLP analysis Total genomic DNAs were isolated from young leaves of individual plants using a modified 2X CTAB method (Doyle and Doyle, 1987). The AFLP method was adapted from Vos et al., 1995 using simultaneous restriction and ligation of DNA sampling.

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Y. Zhang et al. / Scientia Horticulturae 170 (2014) 284–292

Table 1 List of taxa included in this study. The classification of Epimedium follows the system proposed by Stearn, 2002. Species and sample code

Subgenus

Section

Series

Origin

Voucher

1E. campanulatum 2E. campanulatum 3E. platypetalum 4E. platypetalum 5E. ecalcaratum 6E. ecalcaratum 7E. reticulatum 8E. reticulatum 9E. shuichengense 10E. shuichengense 11E. davidii 12E. davidii 13E. hunanense 14E. hunanense 15E. fangii 16E. fangii 17E. fangii 18E. fangii 19E. flavum 20E. flavum 21E. ogisui 22E. pauciflorum 23E. epsteinii 24E. epsteinii 25E. shennogjiaense 26E. mikinorii 27E. mikinorii 28E. pseudowushanense 29E. pseudowushanense 30E. pseudowushanense 31E. wushanense 32E. wushanense 33E. wushanense 34E. ilicifolium 35E. ilicifolium 36E. ilicifolium 37E. jinchengshanense 38E. jinchengshanense 39E. jinchengshanense 40E. jinchengshanense 41E. glandulosopilosum 42E. lishihchenii 43E. lishihchenii 44E. franchetii 45E. franchetii 46E. franchetii 47E. franchetii 48E. franchetii 49E. baojingense 50E. baojingense 51E. enshiense 52E. zhushanense 53E. zhushanense 54E. zhushanense 55E. acuminatum 56E. acuminatum 57E. acuminatum 58E. acuminatum 59E. simplicifolium 60E. chlorandrum 61E. chlorandrum 62E. membranaceum 63E. membranaceum 64E. rhizomatosum 65E. rhizomatosum 66E. leptorrhizum 67E. leptorrhizum 68E. leptorrhizum 69E. leptorrhizum 70E. brachyrrhizum 71E. brachyrrhizum 72E. sutchuenense 73E. sutchuenense 74E. sutchuenense 75E. elongatum 76E. brevicornu

Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium

Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon

Campanulatae Campanulatae Campanulatae Campanulatae Campanulatae Campanulatae Campanulatae Campanulatae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Davidianae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Dolichocerae Brachycerae

China, Sichuan, Dujiangyan China, Sichuan, Dujiangyan China, Sichuan, Wenchang China, Sichuan, Luding China, Sichuan, Baoxing China, Sichuan, Baoxing China, Sichuan, Jinyang China, Sichuan, Jinyang China, Guizhou, Shuicheng China, Guizhou, Shuicheng China, Sichuan, Baoxing China, Sichuan, Baoxing China, Guangxi, Quanzhou China, Guangxi, Quanzhou China, Sichuan, Emei China, Sichuan, Emei China, Sichuan, Emei China, Sichuan, Emei China, Sichuan, Tianquan China, Sichuan, Tianquan China, Sichuan, Lushan China, Sichuan, Maoxian China, Hunan, Sangzhi China, Hunan, Sangzhi China, Hubei, Shennongjia China, Hubei, Enshi China, Hubei, Enshi China, Guizhou, Libo China, Guizhou, Leishan China, Guangxi, Rongshui China, Chongqing, Wushan China China, Hubei, Badong China, Shaanxi, Zhenping China, Shaanxi, Zhenping China, Shaanxi, Pingli China, Sichuan China, Sichuan China, Sichuan China, Sichuan, Tongjiang China, Chongqing, Wushan China, Jiangxi, Lushan China, Jiangxi, Lushan China, Hubei, Shennongjia China, Hubei, Wufeng China, Hubei, Shennongjia China, Hubei, Yichang China, Hubei, Lichuan China, Hunan, Baojing China, Hunan, Baojing China, Hubei, Enshi China, Hubei, Zhushan China, Hubei, Fangxian China, Hubei, Zhushan China, Sichuan, Hongya China, Chongqing, Nanchuan China, Guizhou, Shiqian China, Guizhou, Zhengyuan China, Guizhou, Wuchuan China, Sichuan, Baoxing China, Sichuan, Baoxing China, Sichuan, Dujiangyan China, Sichuan, Dujiangyan China, Sichuan China, Sichuan, Leibo China, Hubei, Hefeng China, Hubei, Lichuan China, Guizhou, Kaili China, Guizhou, Songtao China, Guizhou, Yinjiang China, Guizhou, Yinjiang China China, Hubei, Shennongjia China, Hubei, Shennongjia China China

M. Ogisu 94305 (K) Y.J. Zhang 392 (HIB) D. Probst Cc022263 (US) Y.J. Zhang 380 (HIB) D. Probst Cc970229 (US) Y.J. Zhang 309 (HIB) D. Probst Cc030378 (US) Y.J. Zhang 378 (HIB) D. Probst Cc030200 (US) Y.J. Zhang 375 (HIB) D. Probst Cc950174 (US) Y.J. Zhang 312 (HIB) D. Probst Cc030410 (US) Y.J. Zhang 238 (HIB) Y.J. Zhang 355 (HIB) Y.J. Zhang 356 (HIB) Y.J. Zhang 357 (HIB) Y.J. Zhang 358 (HIB) M. Ogisu 92032 (K) Y.J. Zhang 381 (HIB) D. Probst Cc970227 (US) K.C. Yen & S.L.Shao 66535 (GXMI) D. Probst Cc940255 (K) Y.J. Zhang 188 (HIB) Y.J. Zhang 148 (HIB) M. Ogisu 95039 (K) Y.J. Zhang 056 (HIB) D. Probst Cc021675 (US) D. Probst Cc021471 (US) Q.J. Chen 1973 (HIB) D. Probst Cc014633 (US) D. Probst Cc014633 (US) Y.J. Zhang 010 (HIB) D. Probst Cc93020 (K) Y.J. Zhang 110 (HIB) Y.J. Zhang 113 (HIB) D. Probst Cc012591 (US) D. Probst Cc001827 (US) D. Probst Cc013999 (US) Y.J. Zhang 128 (HIB) Y.J. Zhang 216 (HIB) M. Ogisu 96305 (K) Y.J. Zhang 233 (HIB) Y.J. Zhang 202 (HIB) Y.J. Zhang 191 (HIB) Y.J. Zhang 192 (HIB) Y.J. Zhang 193 (HIB) Y.J. Zhang 337 (HIB) D. Probst Cc030651 (US) Y.J. Zhang 183 (HIB) Y.J. Zhang 021 (HIB) D. Probst Cc022893 (US) Y.J. Zhang 130 (HIB) Y.J. Zhang 117 (HIB) Y.J. Zhang 390 (HIB) Y.J. Zhang 127 (HIB) Y.J. Zhang 125 (HIB) Y.J. Zhang 194 (HIB) D. Probst Cc031511 (US) D. Probst Cc960102 (US) Y.J. Zhang 313 (HIB) D. Probst Cc022185 (US) Y.J. Zhang 391 (HIB) D. Probst Cc980076 (US) Y.J. Zhang 303 (HIB) Y.J. Zhang 195 (HIB) Y.J. Zhang 246 (HIB) Y.J. Zhang 156 (HIB) Y.J. Zhang 175 (HIB) D. Probst Cc940397 (US) Y.J. Zhang 159 (HIB) D. Probst Cc014578 (US) Y.J. Zhang 133 (HIB) Y.J. Zhang 270 (HIB) D. Probst Cc012861 (US) D. Probst Cc013913 (US)

Y. Zhang et al. / Scientia Horticulturae 170 (2014) 284–292

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Table 1 (Continued) Species and sample code

Subgenus

Section

Series

Origin

Voucher

77E. brevicornu 78E. pubescens 79E. pubescens 80E. pubescens 81E. pubescens 82E. pubescens 83E. stellulatum 84E. stellulatum 85E. fargesii 86E. fargesii 87E. qingchengshanense 88E. qingchengshanense 89E. dewuense 90E. dewuense 91E. dewuense 92E. dewuense 93E. dewuense 94E. dolichostemon 95E. dolichostemon 96E. sagittatum 97E. sagittatum 98E. sagittatum 99E. sagittatum 100E. sagittatum 101E. sagittatum 102E. sagittatum 103E. sagittatum 104E. sagittatum 105E. sagittatum 106E. sagittatum 107E. sagittatum 108E. sagittatum 109E. sagittatum var. glabratum 110E. sagittatum 111E. myrianthum 112E. myrianthum 113E. multiflorum 114E. coactum 115E. coactum 116E. coactum var. longtouhum 117E. coactum var. longtouhum 118E. sagittatum var. oblongifoliolatum 119E. borealiguizhouense 120E. borealiguizhouense 121E. truncatum 122E. truncatum 123E. truncatum 124E. elachyphyllum 125E. elachyphyllum 126E. grandiflorum 127E. grandiflorum 128E. grandiflorum 129E. sempervirens 130E. sempervirens 131E. koreanum 132E. koreanum 133E. koreanum 134E. macrosepalum 135E. trifoliolatobinatum 136E. diphyllum 137E. diphyllum 138E. elatum 139E. alpinum 140E. alpinum 141E. pubigerum 142E. pinnatum 143E. pinnatum 144E. perralderianum 145V. hexandra

Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Epimedium Rhizophyllum Rhizophyllum Rhizophyllum

Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Diphyllon Macroceras Macroceras Macroceras Macroceras Macroceras Macroceras Macroceras Macroceras Macroceras Macroceras Macroceras Macroceras Polyphyllon Epimedium Epimedium Epimedium

Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae Brachycerae

China, Shanxi, Qinshui China, Shaanxi China, Shaanxi, Hanzhong China, Shaanxi, Lueyang China, Shaanxi, Foping China, Shaanxi, Taibai China, Hubei, Shiyan China, Hubei, Shiyan China China, Chongqing, Chengkou China, Sichuan, Dujiangyan China, Sichuan, Dujiangyan China, Guizhou, Dejiang China, Guizhou, Dejiang China, Guizhou, Dejiang China, Guizhou, Wuchuan China, Guizhou, Wuchuan China China, Hubei, Lichuan China, Hubei, Luotian China, Fujian, Yongtai China, Guangdong, Ruyuan China, Guangxi, Liuzhou China, Jiangxi, Wuning China, Jiangxi, Nanfeng China, Anhui, Huangshan China, Anhui, Huoshan China, Zhejiang, Kaihua China, Hunan, Liuyang China, Hunan, Cili China, Hunan, Huaihua China, Guizhou, Zhenyuan China, Guizhou, Jiangkou China, Chongqing, Wanxian China, Hunan, sangzhi China, Hunan, sangzhi China, Guizhou, Yuping China, Guizhou, Jianhe China, Guizhou, Taijiang China, Guizhou, Kaili China, Guizhou, Kaili China, Hubei, Enshi China, Guizhou, Yanhe China, Guizhou, Yanhe China, Hunan, Baojing China, Hunan, Zhangjiajie China, Hunan, Baojing China, Guizhou, Songtao China, Guizhou, Songtao Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Kashmir South Europe South Europe Caucasia Iran Iran North Africa American

Y.J. Zhang 356 (HIB) D. Probst Cc022557 (US) Y.J. Zhang 321 (HIB) Y.J. Zhang 107 (HIB) Y.J. Zhang 044 (HIB) Y.J. Zhang 322 (HIB) D. Probst Cc960139 Y.J. Zhang 122 (HIB) D. Probst Cc001630 Y.J. Zhang (HIB) D. Probst Cc013017 (US) Y.J. Zhang 393 (HIB) D. Probst Cc020548 (US) Y.J. Zhang 163 (HIB) Y.J. Zhang 249 (HIB) Y.J. Zhang 165 (HIB) Y.J. Zhang 171 (HIB) D. Probst Cc011987 (US) Y.J. Zhang 247 (HIB) Y.J. Zhang 252 (HIB) X.J. Zhang & Y.Q. Xu 27 (HIB) X.J. Zhang & Y.Q. Xu 30 (HIB) Y.J. Zhang 257 (HIB) X.J. Zhang & Y.Q. Xu 20 (HIB) X.J. Zhang & Y.Q. Xu 16 (HIB) X.J. Zhang & Y.Q. Xu 33 (HIB) Y.J. Zhang 254 (HIB) X.J. Zhang & Y.Q. Xu 35 (HIB) X.J. Zhang & Y.Q. Xu 38 (HIB) X.J. Zhang & Y.Q. Xu 41 (HIB) X.J. Zhang & Y.Q. Xu 13 (HIB) X.J. Zhang & Y.Q. Xu 23 (HIB) Y.J. Zhang 088 (HIB) Y.J. Zhang 250 (HIB) D. Probst Cc940019 (US) Y.J. Zhang 185 (HIB) Y.J. Zhang 148 (HIB) Y.J. Zhang 196 (HIB) Y.J. Zhang 398 (HIB) D. Probst Cc021384 (US) Y.J. Zhang 158 (HIB) Y.J. Zhang 020 (HIB) D. Probst Cc020708 (US) Y.J. Zhang 231 (HIB) D. Probst Cc030564 (US) Y.J. Zhang 343 (HIB) Y.J. Zhang 296 (HIB) D. Probst Cc031443 Y.J. Zhang 178 (HIB) D. Probst Cc950055 (US) D. Probst Cc950058 (US) D. Probst Cc950057 (US) D. Probst Cc920030 (US) D. Probst Cc950036 (US) D. Probst Cc970079 (US) D. Probst Cc013228 (US) D. Probst Cc970145 (US) D. Probst Cc03s.n. (US) D. Probst Cc950046 (US) from Cornell University D. Probst Cc940544 (US) provided from B.L. Guo from Cornell University D. Probst Cc920002 (US) D. Probst Cc950215 (US) D. Probst Cc890003 (US) D. Probst Cc950217 (US) D. Probst Cc980084 (US) D. Probst Cc950015 (US)

The pre-amplification was performed with EcoRI/Msel pairs, each containing one selective nucleotide (EcoRI + A, Msel + C). Final amplifications were performed with two or three selective nucleotides on each primer, and 270 primer combinations were screened and checked. We selected eight primer combinations

for final AFLP analysis (Table 2). Gels were stained with silver nitrate (Bassam et al., 1991). Fragments were scored in a length range from 50 to 300 base pairs. AFLP patterns were compiled into a single binary data matrix (presence = 1, absence = 0) for final analysis.

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Y. Zhang et al. / Scientia Horticulturae 170 (2014) 284–292

Table 2 AFLP primer combinations and numbers of fragments scored for 144 individuals from Epimedium and from one individual of Vancouveria hexandra, as outgroup. Number of fragments Selective primer combination

Variable

Primer pair EcoRI-A MseI-C Scored Across all taxa

Within Epimedium

1 AGC CGA AGT CAG 2 3 ACG CAC 4 AAC CGT AGG ACC 5 ACT CAC 6 AGG CGT 7 AAC ACC 8 All primer combinations

67 67 91 74 61 55 18 73 506

70 75 98 80 67 62 19 78 549

67 67 91 75 61 58 18 74 511

2.3. Data analysis The AFLP data set was analyzed with MrBayes v.3.1 (Ronquist and Huelsenbeck, 2003) using the restriction site (binary) model. This analysis consisted of two simultaneous Markov Chain Monte Carlo (MCMC) runs with four chains and 20 million generations, sampling every 2000 generation, a heating parameter value of 0.2. The first 25% trees were discarded as burn-in, while the remaining trees were used to construct the 50% majority rule consensus tree and to estimate the posterior probabilities (PP). 3. Results Using eight primer combinations, we scored 549 AFLP fragments, 511 of which were polymorphic across the full set of taxa. Within Epimedium, 506 characters were polymorphic. Table 2 lists the total number of polymorphic fragments scored from different primer pairs, which varied from 18 to 91 across all taxa. All accessions had unique AFLP profiles. The phylogenetic relationships among Epimedium species are depicted in Fig. 2. In the dendrogram, Epimedium was separated into two monophyletic clades, subgen. Rhizophyllum and subgen. Epimedium. Within subgen. Epimedium, all four sections formed well-supported monophyletic clades (sect. Diphyllon, PP100; sect. Macroceras, PP100; sect. Epimedium, 86). Sect. Diphyllon firstly clustered with E. elatum (sect. Polyphyllon) (PP 90), and then formed a trichotomy with sect. Macroceras and sect. Epimedium. For the sect. Diphyllon clade, 47 Chinese Epimedium species were subdivided into five well-supported monophyletic clades related to their floral characteristics except that five species were either isolated or formed a general polytomy. Clade-1 contained four species from ser. Campanulatae, 12 species from ser. Davidianae, and eight species from ser. Dolichocerae (PP 100). Within clade-1, the accessions of each species (except E. platypetalum, E. franchetii, and E. baojingense) formed a monophyletic cluster. Furthermore, all the four species of ser. Campanulatae clustered with five species of ser. Davidianae (PP 64). Clade-2 comprised 30 accessions of seven species from ser. Brachycerae (PP 98). E. sagittatum #108, #96, and #103 were early separated from the rest, and the remaining accessions were subdivided into four subclusters: (I) E. truncatum and E. elachyphyllum were united in a well-supported clade (PP 92); (II) E. borealiguizhouense, E. sagittatum var. oblongifoliolatum, E. myrianthum, and E. sagittatum var. glabratum, all from northeastern Guizhou, northwestern Hunan, and western Hubei; (III) E. sagittatum all from eastern and central China except #110 from Chongqing; and (IV) E. multiflorum, E. coactum, E. coactum var. longtouhum, and E. sagittatum (#99, #107), all from western Hunan, eastern Guizhou, and northern Guangxi (PP 88).

Clade-3 was composed of four species from ser. Brachycerae (PP 97). Among these four species, the seven collections of E. dolichostemon and E. dewuense clustered into a clade (PP 80), as did the four accessions of E. fargesii and E. qingchengshanense (PP 97). Clade-4 contained five species from ser. Dolichocerae (PP 99). The seven collections of E. acuminatum, E. simplicifolium, and E. chlorandrum together formed a clade (PP 100), in which all the collections from Chongqing and Guizhou formed a subclade (PP 82) while those from Sichuan fell into other subclade (PP 100). The remaining two species, E. membranaceum and E. rhizomatosum, formed a strongly supported cluster (PP 100). Clade-5 only possessed two species of ser. Dolichocerae, E. leptorrhizum, and E. brachyrrhizum (PP 89). All these six accessions of the two species were separated into two sister clusters: one comprised E. leptorrhizum #66 and #67 from western Hubei (PP 100), while the other included E. leptorrhizum and E. brachyrrhizum from Guizhou (PP 94). The remaining five species of sect. Diphyllon were not found within these clades, and comprised E. sutchuenense and E. elongatum from ser. Dolichocerae, and E. brevicornu, E. pubescens, and E. stellulatum from ser. Brachycerae. Of these, E. stellulatum was isolated while the other four species formed a general polytomy. Furthermore, the accessions of each of E. pubescens, E. brevicornu, and E. sutchuenense did not cluster into monophyletic group. 4. Discussion 4.1. Taxonomic evaluation of subgenera and sections within Epimedium Stearn, 2002 classified Epimedium into two subgenera and four sections. Consistent with previous molecular phylogenetic studies (Zhang et al., 2007; Smet et al., 2012), the present AFLP data (Fig. 2) suggested that subgen. Rhizophyllum and four sections of subgen. Epimedium grouped into five distinct clusters. Furthermore, our results for the first time supported Stearn’s (2002) subgeneric circumscription of Epimedium albeit with low PP. Epimedium is a member of basal eudicots with a disjunctive distribution in Mediterranean region and eastern Asian, and it reaches its greatest species diversity in central-southeastern China (Ying, 2002). In the updated system of Epimedium (Stearn, 2002), subgen. Rhizophyllum and the four sections of subgen. Epimedium were closely correlated with their geographical distribution. The phylogenetic studies robustly supported the sister relationship between sect. Macroceras and sect. Epimedium based on ITS and atpB-rbcL spacer sequences (Zhang et al., 2007). However, the other relationships between subgen. Rhizophyllum and four sections of subgen. Epimedium were unresolved or poorly supported based on different molecular markers including the present AFLP analyses (Sun et al., 2005; Zhang et al., 2007; Smet et al., 2012). Although Zhang et al., 2007 estimated divergence times of the major clades of Epimedium, it still remains unresolved in origin, evolution, migration, and dispersal of the genus in North Temperate Zone. Thus, more effective molecular markers are needed to analyze the phylogenetic relationships among the major clades of Epimedium, to evaluate the scheme of Stearn, 2002, and to investigate biogeography of the genus. 4.2. Phylogenetic relationships within Chinese Epimedium of sect. Diphyllon Stearn, 2002 classified Chinese Epimedium of sect. Diphyllon into four series based on flower morphology. Ser. Campanulatae bears small and campanulate flowers with flat petals or with a slight nectarial swelling at base (Fig. 1A, B). Ser. Davidianae possesses large flowers with petal laminae that expand with an elongated curved spurs (Fig. 1C, D). Ser. Dolichocerae has large flowers with

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Fig. 2. The 50% majority rule consensus tree resulting from Bayesian inference for AFLP data, showing the phylogenetic relationships among Epimedium species. Posterior probabilities are indicated above the relevant branches.

petals bearing long spurs without basal laminae (Fig. 1E–G). Ser. Brachycerae has relatively small flowers and very short petals that are saccate or with short spurs (Fig. 1H-L). Higher resolution of phylogenetic relationships within sect. Diphyllon, however, has not been provided based on pollen types, flavonoid types, karyotypes, and molecular markers (Guo et al., 1998, 2008; Guo and Xiao, 1999; Sun et al., 2005; Shen et al., 2007; Zhang et al., 2007,

2008; Sheng et al., 2010, 2011b; Smet et al., 2012). The present AFLP data provided clear evidence for the first time on the phylogenetic relationships among 47 species in sect. Diphyllon. Bayesian dendrogram analysis (Fig. 2) subdivided sect. Diphyllon into five well-supported monophyletic clades related to the floral characteristics except that five species were either isolated or formed a polytomy.

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Clade-1 contained 24 species from ser. Campanulatae, ser. Davidianae, and ser. Dolichocerae, morphologically characterized by flat petals or petals with long spurs (Fig. 1A-E, Fig. 2). Within this clade, all four species of ser. Campanulatae and five species of ser. Davidianae formed a subgroup, and are characterized by being small with ovate leaflets and are derived from western Sichuan and Guizhou. The close relationship between these two series is consistent with previous molecular phylogenetic studies, albeit with much fewer collections (Zhang et al., 2007; Smet et al., 2012). The remaining 15 species within clade-1 belonged to ser. Davidianae and ser. Dolichocerae. Among these, only E. pauciflorum shares similar morphological characters and distribution with the cluster mentioned above. The other 14 species are much sturdier and bear ovate or lanceolate leaflets. Furthermore, these species are concentrated in eastern Sichuan and Guizhou, western Hunan and Hubei, Chongqing and southern Shaanxi (except E. lishihchenii from western Jiangxi). In addition, E. pauciflorum and five of these 14 species (E. franchetii, E. zhushanense, E. ilicifolium, E. baojingense, and E. enshiense) grouped into a cluster. Except for E. pauciflorum, these five species belong to ser. Dolichocerae and possess highly similar large flowers. Clade-2, a sister clade to clade-1 with high posterior probabilities (PP 99), comprised seven species from ser. Brachycerae. These are distinctly characterized by having flowers smaller than 12 mm in diameter with 2-4-mm-long saccate petals (Fig. 1L, Fig. 2), and covered all the species with the smallest flowers in ser. Brachycerae. Clade-3 was composed of four species from ser. Brachycerae, E. dolichostemon, E. dewuense, E. fargesii, and E. qingchengshanense (Fig. 1H, I, Fig. 2). These four species have small flowers slightly larger than those of clade-2, whose petals bear spurs much shorter than inner sepals. Furthermore, these are the only four species within sect. Diphyllon to contain conspicuously protruded stamens, 8-10 mm long. Clade-4 and clade-5 included seven species of ser. Dolichocerae (Fig. 1F, G, Fig. 2), which share large flowers bearing long spurred petals without basal laminae. The remaining included five species from ser. Dolichocerae and ser. Brachycerae. These species were either isolated or formed a general polytomy, and it was paraphyletic for the accessions of each of E. pubescens, E. brevicornu, and E. sutchuenense. Likewise, it was paraphyletic for the intra-species accessions of E. platypetalum, E. baojingense, and E. franchetii (clade-1 of Fig. 2). It needs further molecular phylogenetic investigation on these species combining morphological analyses. Chinese Epimedium species showed selfincompatible and interfertile with highly uniform karyotype, and some hybrids have been reported (Stearn, 2002; Zhang et al., 2008; Sheng et al., 2011a). The observed paraphylies within intra-species accessions could be caused by reticulate evolution in Chinese Epimedium. In general, the present subdivision of Chinese sect. Diphyllon was well supported and correlated with floral morphology, and partly supported the previous dissection of four series (Stearn, 2002). However, the results provided less information on the relationships among the five main clades of the section. It needs more effective molecular markers to further investigate the phylogeny and evolution of Chinese sect. Diphyllon. 4.3. Taxonomic implication of AFLP analysis of Chinese Epimedium of sect. Diphyllon Chinese Epimedium of sect. Diphyllon presents a number of taxonomic controversies (Guo et al., 2008; Ying et al., 2011; Zhang et al., 2011; Smet et al., 2012), but no molecular data can successfully resolve its taxonomy. In the present AFLP analysis, Bayesian dendrogram demonstrated that accessions from the same Chinese Epimedium species (but a few species) formed a clade consisting of subclades mainly

correlated with geographical distribution. Species with similar morphology, particularly floral characteristics, were grouped and related to geographical distribution. These results shed new light into the taxonomy of the questionable Chinese Epimedium species. E. reticulatum is a disputable species in the flower morphology (Bao, 1987; Guo et al., 2008; Ying et al., 2011), which was classified into ser. Brachycerae and ser. Dolichocerae by Stearn, 2002 and Guo et al., 2008. The morphological studies recognized E. reticulatum with cucullate and spurless petals, and placed the species into ser. Campanulatae (Zhang et al., unpublished data). The AFLP approach nested E. reticulatum in the clade containing species of ser. Campanulatae and ser. Davidianae (clade-1 of Fig. 2), consistent with the results of the morphological taxonomy. Recent taxonomical studies showed that the protologue of E. wushanense was in fact the mixed description of three distinct Epimedium species, E. wushanense, E. ilicifolium, and E. jinchengshanense, which are somewhat similar in leaf shapes (Zhang et al., unpublished data). The AFLP profiles separated the accessions of these three species into three distinct branches (clade-1 of Fig. 2) that can be easily distinguished by the morphological characteristics of their flowers, inflorescences, and capsules. E. wushanense belongs to ser. Davidianae which possesses petals with obvious basal laminae, compact inflorescences, and short and fat capsules, while E. ilicifolium and E. jinchengshanense belong to ser. Dolichocerae and both have petals lacking basal laminae, loose inflorescences, and relatively long and slender capsules. Additionally, E. ilicifolium and E. jinchengshanense can be separated from each other by the thickness of subulate petals and the size of inner sepals. E. dewuense was published only based on the comparison with E. sagittatum, but not with E. dolichostemon (He and Xu, 2003). According to the protologue of E. dolichostemon and E. dewuense, the two species were only distinguished by the shape and pubescence of their leaflets (Stearn, 1990; He and Xu, 2003). Based on the investigation in the herbaria and the fields, Zhang et al. (unpublished data) found that the morphological variations of leaflets between the two species were continuous, and thus reduced E. dewuense as a synonym of E. dolichostemon (Fig. 1I). The AFLP data indicated that all the accessions of these two species mixedly grouped into a distinct cluster (clade-3 of Fig. 2), which supported the recent taxonomic treatment. All the accessions of E. acuminatum, E. simplicifolium, and E. chlorandrum mixedly clustered into a separate clade (Fig. 1F,G, clade-4 of Fig. 2). E. simplicifolium was reported based on distinguishing from E. acuminatum by having unifoliolate leaves with sericeous hairs (Ying, 1975). However, the leaves of E. acuminatum may also be unifoliolate or have sericeous hairs, and E. simplicifolium was reduced into synonym (Zhang et al., 2011). E. chlorandrum differed from E. acuminatum by its inner sepals being slightly ascending and not closely appressed to the petals and by its anthers and pollen being green (Stearn, 1997). Zhang et al. (unpublished data) found the diagnostic features of E. chlorandrum fell into the variation range of E. acuminatum and treated it as a synonym. So the present AFLP analyses also confirmed the classical treatment on the three species. Smet et al., 2012 classified leaflet pubescence of Epimedium into four types, and reported that these types formed clear-cut, discrete character states. However, the leaflet pubescence of Epimedium is very complex. Within one Epimedium species, such as E. acuminatum or E. sagittatum, there are many types of leaflet pubescence and the variations are continuous among individuals and populations (Zhang et al., 2011; Xu et al., 2013). Compared to leaf pubescence, floral character is much more stable as reproductive organ although it appears variations in some species. As a result, it needs intensive variation investigations to identify the boundaries of Epimedium species.

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E. leptorrhizum and E. brachyrrhizum shared similar morphological characters. The major difference between the two species was originally as being that E. leptorrhizum had elongated slender rhizomes while E. brachyrrhizum bore more compact, clump-forming rhizomes (Stearn, 1997). However, examination of a series of E. leptorrhizum specimens showed its rhizomes were often slender and long-creeping but occasionally thicker and spreading more slowly (Zhang et al., unpublished data). In the present AFLP analysis, the accessions of the two species mixedly clustered into a distinct clade (clade-5 of Fig. 2), so it was reasonable to treat E. brachyrrhizum as a synonym of E. leptorrhizum. E. sagittatum and its related species are the most controversially defined in Epimedium taxonomy (He et al., 2003; Ying et al., 2011; Xu et al., 2013), and are referred to as E. sagittatum complex here. This E. sagittatum complex includes five species and three varieties (E. sagittatum, E. myrianthum, E. borealiguizhouense, E. multiflorum, E. coactum, E. sagittatum var. glabratum, E. coactum var. longtouhum, E. sagittatum var. oblongifoliolatum), and possesses greatest variation and distribution in China (from eastern China, across central China to southwest China). The present AFLP analyses revealed that the complex was subdivided in a manner closely correlated with geographical distribution. For the E. sagittatum complex, the taxa from central and eastern China (subclade III of Fig. 2 excluding E. sagittatum #110), from western Hubei, northwestern Hunan, and northeastern Guizhou (subclade II of Fig. 2) and from western Hunan, eastern Guizhou, and northern Guangxi (subclade IV of Fig. 2) formed three clusters. The taxa of subclade III (again excluding E. sagittatum #110) have relatively consistent morphological characteristics typical of E. sagittatum, including narrow and straight inflorescence with petals of similar length as inner sepals. The taxa of subclade II and IV possess broader inflorescences with petals that are much shorter than inner sepals. Furthermore, within subclade II, E. borealiguizhouense and E. sagittatum var. oblongifoliolatum have highly similar morphological characters and formed a strongly supported cluster (PP 99). These two taxa have lanceolate leaflets that distinguish them from the other taxa of subclade II and IV, which have ovate leaflets. In addition, E. sagittatum #110 from Chongqing is distinguished with other accessions of subclade III by its inflorescence and leaflet morphologies. Three accessions of E. sagittatum (#96, #103, #108) did not group within the three subclusters, which needs further investigations. In general, E. sagittatum complex has great morphological variations with widest geographical distribution, and needs further research on taxonomy and phylogeny based on more extensive field investigation and more valid molecular marker analyses. 5. Conclusion AFLP markers have been employed successfully to examine the relationships between Epimedium species, especially Chinese species. The results offer a solid base for the identification of Epimedium species, which will lead to more efficient utilization of the natural germplasm of this important horticultural and medicinal genus. Acknowledgement This research was financially supported by National Natural Science Foundations of China (Nos. 30900076, 31100146) and Key Research Program of the Chinese Academy of Sciences (KSZD-EWZ-004). We are grateful to Mr. Darrell Probst, Pro. Baolin Guo and Botanical Garden of Cornell University for providing leaf materials of Epimedium and Vancouveria species. We thank Dr. John Lonsdale for providing the picture of E. ecalcaratum. We also give our sincere thanks to Prof. Dan Binkley, Jianqiang Li, Baolin Guo, Xinwei Li,

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Tieyao Tu, and Dr. Alice Hayward for their constructive comments on our manuscript.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta.2014. 02.025.

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