The taxonomic relevance of flower colour for Epimedium (Berberidaceae), with morphological and nomenclatural notes for five species from China

Abstract Morphological variations, particularly flower colour, could be considered as an evolutionarily and ornamentally significant taxonomic criterion for Epimedium. Our extensive field investigation based on population studies revealed abundant intraspecific variations in flower colour. Five species, (i.e., E.acuminatum Franch., E.leptorrhizum Stearn, E.pauciflorum K.C.Yen, E.mikinorii Stearn, and E.glandulosopilosum H.R.Liang) were found to possess polymorphic flower colour, which is first described and illustrated here. Moreover, all these species were found to be polymorphic in other diagnostic characters, such as the type of rhizome, the number and arrangement of stem-leaves, and/or their indumentum, which have not been adequately described in previous studies. Therefore, their morphological descriptions have been complemented and/or revised. We also provide notes on the morphology and nomenclature for each species. Additionally, a key to the species in China has been provided. The present study could serve as a basis for understanding their taxonomy and helping their utilisation as an ornamental plant.


Introduction
Epimedium L. is the largest herbaceous genus of Berberidaceae, comprising ca. 60 species distributed in the temperate mountain regions from eastern Asia to northwestern Africa, with enormous distributional gaps within these regions (Stearn 2002;Xu et al. 2014a;Zhang et al. 2015). China represents the diversity centre for the genus, with ca. 50 endemic species (except E. koreanum Nakai) (Stearn 2002;Ying 2002). Epimedium is an attractive genus, both horticulturally and botanically, and has received increased attention in past decades (Avent 2010;Ward 2004;Ying 1975Ying , 2001Ying , 2002Stearn 2002;Zhang et al. 2011Zhang et al. , 2014Zhang et al. , 2015Liu et al. 2016Liu et al. , 2017. Since the 1980's, further botanical exploration and collections in China have dramatically increased the number of Epimedium species in this country (Ying 1975;Ying et al. 2011;Stearn 2002;Xu et al. 2014a). However, many Epimedium species have been described for China based on a single locality and/or the descriptions were based on limited specimens or collections (Stearn 1997;Zhang et al. 2011Zhang et al. , 2014Xu et al. 2014a). Due to the lack of additional field investigation and observation of morphological characters, the morphological variation amongst many Epimedium species is not yet well understood (Xu et al. 2014a(Xu et al. , 2014bZhang et al. 2015;Liu et al. 2016Liu et al. , 2017. From 1990 to 1998, Stearn used a small collection of Epimedium to describe 16 species, which were based on a limited number of cultivated specimens (Stearn 1990(Stearn , 1993a(Stearn , 1993b(Stearn , 1995(Stearn , 1996(Stearn , 1997(Stearn , 1998, and thus, the morphological descriptions of some species were incomplete and/or inaccurate (Buck 2003;Xu et al. 2014a;Zhang et al. 2015;Liu et al. 2016). Our recent studies have focused on the standardisation of morphological descriptions in the genus (e.g., Liu et al 2016Liu et al , 2017. The divergence of floral traits is a striking phenomenon in flowering plants, which plays an important role in ecology, plant systematics and conservation (Vaidya et al. 2018). Specifically, flower colour has been demonstrated to be an important feature for identifying and classifying species (Ying 2001;Stearn 2002;Ying et al. 2011). Epimedium has the greatest range of flower colour than any other genus of Berberidaceae, varying from white (e.g., E. latisepalum Stearn) and yellow (e.g., E. ecalcaratum G.Y.Zhong, E. fangii Stearn, E. flavum Stearn, and E. franchetii Stearn) to pink (e.g., E. leptorrhizum Stearn), and to purple (e.g., E. epsteinii Stearn, E. pseudowushanense B.L.Guo, and E. zhushanense K.F.Wu & S.X.Qian) (Stearn 2002;Ying et al. 2011). The flower colour variation could be evolutionarily significant for Epimedium. However, compared to interspecific variation, intraspecific variation in flower colour is relatively uncommon (Matsumura et al. 2006). Usually, the colour of flowers is constant at the species level, but E. grandiflorum C.Morren and E. acuminatum Franch. are believed to be heterochromic (Stearn 2002;Ying et al. 2011). Epimedium grandiflorum varies greatly in flower colour from white to pale yellow or light purple to reddish-purple to deep pink or violet (Stearn 2002). For E. acuminatum, intraspecific colour variation from white to yellow to pale violet or purple has been reported sporadically (Ying et al. 2011;Zhang et al. 2011Zhang et al. , 2015. However, systematic investigations of flower colour variation are scarce for Epimedium, with the taxonomic significance or mechanism of the variation currently unknown. The flower colour diversity may be a signal of plant speciation, which also may be one of the reasons for the difficulty in assessing it for taxonomy (Bradshaw et al. 1995;Matsumura et al. 2006;Hopkins and Rausher 2011). Therefore, the study of the flower colour variation is of great significance to the taxonomy ranking of Epimedium, as well as for its evolution (Wang et al. 2017).
As part of our ongoing efforts to study the Epimedium diversity in China, we studied the relevance of flower colour variation for the taxonomy of Epimedium based on extensive field studies for five species of Chinese Epimedium, with additional cultivation and herbaria studies. Moreover, we present a comprehensive revision on their taxonomic description, including the variation on rhizome morphology, the number and arrangement of stem-leaves, and indumentum type. Additionally, we provide an identification key for the species of Epimedium recognised in China. The results provide abundant and important information for the taxonomy of Epimedium, and subsidies in its exploration and utilisation, for example, as ornamental plants.

Materials and methods
In Epimedium, some diagnostic features, especially those of flowers and inflorescences, are obscured or not visible in the herbarium material. Moreover, the flowers are of frail texture and deciduous. Therefore, it is difficult to infer the exact colouration of in vivo flowers by examining dried specimens. Therefore, the vegetative as well as reproductive characters were examined mainly in their natural habitats, while the herbarium specimens were used as aids. Herbarium specimens were examined from the following herbaria: CDBI, E, GXMI, HGAS, HIB, IBK, IBSC, IMD, JXCM, K, KUN, N, NAS, P, PE, and SM (acronyms according to Thiers, continuously updated).
All field investigations and observations were conducted in full bloom from 2012 to 2017. To investigate the morphological variation, we carried out field surveys throughout the Chinese distribution range of Epimedium, especially in Sichuan, Guizhou, Guangxi, Hubei, Hunan, Anhui and Jiangxi provinces and Chongqing municipality. In each province or municipality, a survey, as wide and thorough as possible, was conducted. The species that exhibited variations in flower colour and other characters were then observed and sampled (Table 1). Herbarium specimens collected during our fieldwork were deposited in the herbarium of Jiangxi University of Traditional Chinese Medicine (JXCM). The ecological information of the relevant populations was recorded. With the aim of confirming the identifications and better understanding the difference between individuals, as well as populations, 20-30 individuals were collected from each population and posteriorly transplanted to the Jiangxi University of Traditional Chinese Medicine for further observations and studies. Quantitative measurements of rhizome diameter, the number of stem-leaves, height of the flowering stem, length of inflorescence, and the number of flowers were recorded for each specimen. Simultaneously, the following discrete morphological characters were also observed: type of rhizome; indumentum; arrangement of stem-leaves; inflorescence *: the population with flower colour variation morphology; variation of flowers (i.e., shape, colour and proportion); and the colour of pollen in vivo. In addition, particular attention was paid to preparing herbarium specimens, deposited at JXCM herbarium, and photographing plants and also their floral parts in their natural habitat. The Flora of China (Ying et al. 2011) and the monograph of the genus Epimedium (Stearn 2002) were followed to describe the vegetative and reproductive characters of the studied species.

Epimedium acuminatum
Distribution and habitat. Epimedium acuminatum is one of the most widespread species in the genus, distributed in Sichuan, Guizhou, Chongqing, and northern Yunnan. Its large distribution area is predominantly characterised by mountain land. Epimedium acuminatum is often found on mountain slopes, forest edges or weedy slopes with elevations ranging from 270 m to 2400 m (Fig. 2).
Phenology. Epimedium acuminatum flowers from April to June, and fruits from May to July.
Taxonomical remarks.  Zhang et al. 401131), and white (K. J. Guan et al. 477,K. Y. Lang 3002,K. J. Guan et al. 165) or whitish (Z. X. Qu 1057, Sanxia Exped. 0729 and Sanxia Exped. 0909) were the most frequently recorded flower colours. However, specific colours, for example, reddish (S. P. Pong 6108), yellowish purple (X. Y. He 4050; T. C. Pan & G. F. Wu 105), and pale purple-green (Z. Y. Wu 60) have also been examined from specimens. Colour differences among individuals of the same location have been slightly recorded, but both yellow and purple flowers were recorded in Sichuan Econ. Pl. Exped. 0013. Moreover, continuous variations from yellow to white and from yellow to pale reddish-purple were remarked by T. H. Tu 2763 and B. L. Guo 0608, respectively.
Two synonyms are included in E. acuminatum, namely E. simplicifolium and E. chlorandrum. There were only two specimens of E. simplicifolium for reference. The holotype (P. C. Tsoong 606) recorded yellow flowers with purplish red petals. Since these descriptions were based on flowers that are not fully open -the outer sepals soon falling -the outermost is formed of inner sepals. Therefore, the "yellow flower" actually is "yellow or yellowish inner sepals". Another specimen (S. Z. He 96410) recorded a purplish red flower, and the inner sepals spotted with purplish red. Epimedium chlorandrum has six specimens. Greenish inner sepals and pale yellow petals have been described in the holotype (Ogisu 94003). Pale yellow inner sepals and petals were recorded in B. L. Guo 0540 and B. L. Guo 0607 while B. L. Guo 0606 described the colour variation of inner sepals as anything from pale yellow to pale purplish red. And B. L. Guo 0608 also described both inner sepals and petals from pale yellow to pale purplish red.
Based on a field survey at the population level, we observed more extensive and continuous colour variation from pale yellow to dark purple (Fig. 1). Pure white flow- ers have not been observed, but the pale yellow and whitish are very close to white. We therefore speculate that the white flowers described in the specimens might represent pale yellow or whitish flowers instead. Regarding the yellow and purple flowers, there was abundant colour variation among populations as well as among individuals. For example, yellow ranged from pale yellow to yellow, while purple ranged from pale violet or reddish-purple to purple and dark purple. Moreover, there were also many transitions between yellow and purple.
For E. acuminatum, the populations that showed uniform colour (yellow or purple) were excluded from the illustration in this study. We mainly focused on the populations that presented colour variation (Fig. 1). For example, SCMP showed mainly  yellow flowers; only one out of the 30 individuals presented diverse colour, with a yellow spur tinged with a ray of rose inside the base of the petals. SCSL mainly had yellow flowers; parts of individual flowers showed pale yellow, and one individual presented purple-yellow at the base of the petals. CQNC primarily displayed yellow flowers; several individuals revealed a rose margin at the base of the petals, and one individual revealed reddish-purple flowers. SCSS was the most special population with 20 individuals had purple flowers while the rest (10 individuals) had yellow flowers. Moreover, SCSS was the only population for which the colour showed a continuous variation from yellow to purple. The flowers of SCEM and SCYJ were mainly with purple flowers, and purple-yellow occasionally (Fig. 1). In the above populations, SCMP and SCSS were E. chlorandrum.
Combing the geographical distribution of specimens (both field and herbarium specimens) and their flower colour variation of E. acuminatum, we found an interesting result (Fig. 2). Geographic variation in flower colour pattern within E. acuminatum showed a north-south geographic trend. The specimens from the northern area of its distribution mainly have yellow flowers, while the southern ones usually have purple flowers. The polymorphism of flower colour is mainly concentrated in the northwest of its distribution area.
Distribution and habitat. Epimedium leptorrhizum is distributed in the montane forests or thickets in Guizhou, Hubei, Hunan and Chongqing, in elevations ranging from 350 m to 2100 m (Fig. 4). Phenology. Epimedium leptorrhizum flowers from April to May, and fruits from May to June.
Taxonomical remarks. Abundant and continuous flower colour variation has been observed and illustrated for this species. The inner sepals presented continuous variation from white, white tinged with rose, through to completely rose. Petals varied from white or pale yellow with base yellow or orange, white with base rose or deep rose to completely rose, deep rose or pale purple (Fig. 3). Among them, white and pale yellow were only observed in one individual of the GZGY population. However, it is very common that the colour ranged from pale rose or rose to deep rose or pale purple among and within populations. The synonym, E. brachyrrhizum (GZLC) didn't show any variation of flower colour, with rose inner sepals and pale rose petals.
Distribution and habitat. Epimedium pauciflorum is only known from the mountains of Maowen (the holotype locality, once and now divided into Maoxian, Wenchuan and Lixian) in Sichuan province, usually occurring in forest edges and weedy slopes, at high elevations approximately 1700-2600 m (Fig. 4).
Phenology. Epimedium pauciflorum flowers from April to May, and fruits also from April to May.
Taxonomical remarks. Epimedium pauciflorum is a low-growing species with a few-flowered inflorescence. Some information about the colour variation was recorded in part of the specimens. The colours of three specimens (Z. L. Kun 89022, B. L. Guo & W. K. Bao 97040,B. L. Guo 88182) were described as white, while the other two (B. L. Guo & W. K. Bao 97031 and 97032) were described as yellow. We observed a variety of colour variations; the inner sepals were white, faintly purple-tinged or pale rose, whereas the petals were white, white tinged with pale rose, rose, pale yellow, pale rose with the base purple yellow (Fig. 5). Among them, the pale-yellow flowers were only observed in SCJZ population. The specimens (B. L. Guo & W. K. Bao 97031 and 97032) that were with yellow flowers and the SCJZ population in this study were sampled from the same location.
All the examined specimens were with 1 trifoliolate leaf on the flowering stem uniformly. But 1 unifoliolate leaf (only in SCWC population) or 2 alternate trifoliolate leaves (only in SCJZ population) (Fig. 5) were also observed in our field investigation.
Distribution and habitat. Epimedium mikinorii is restricted to the mountains of Hubei (Enshi), Western of China, usually occurring at elevations ranging from 500 m to 1700 m (Fig. 4).
Phenology. Epimedium mikinorii flowers from April to June, and fruits from May to June.
Taxonomical remarks. The field investigation found extensive colour variations. The inner sepals have different colours, ranging from white, rose-tinged, purple-tinged, through to rose and pale purple. Petals also exhibited abundant colour variation, such as yellow, orange, purple, purple but laminae yellow-edged or yellow at both ends while purple in the middle. Although the specimens of E. mikinorii that can be referred to are very limited, the characters and the descriptions of some herbarium specimens were consistent with our observations. Some specimens from Enshi had yellow flowers (B. L. Guo  According to our field investigation and the common garden experiment on two populations, parts of the spurs were almost straight, while most were slightly curved (Fig. 6). The petals, in fact, presented a continuous variation from much longer to slightly longer than the inner sepals (Fig. 6).
As far as the indumentum of leaves concerned, we observed that approximately 40% of the individuals from HBES (near its type locality) and some individuals from HBXT were with white appressed hairs abaxially. Moreover, all the previous descrip- tions and specimens on the flowering stem of E. mikinorii were with 2 opposite trifoliolate leaves. However, 3-whorled trifoliolate was also observed in the HBES population (S. X. Liu et al. 2016017).
Distribution and habitat. Endemic to Chongqing, Western of China, usually occurs in forests or thickets. The elevations ranged from 850 m to 1160 m (Fig. 4).
Phenology. Epimedium glandulosopilosum flowers from April to May, and fruits from May to June. Taxonomical remarks. The observations of the present study showed that only several individuals had yellowish flowers, while the rest had pale purple or purple flowers (Fig. 7). Only two specimens of E. glandulosopilosum are available for reference, but the specimens showed similar results. B. L. Guo & X. Z. Luo 89003 described the flower colour as whitish to pale purple, rarely yellow, while B. L. Guo A15 described it as pale purple or pale yellow.
In the present study, all individuals from the population CQWX (Wuxi) were with long creeping and slender rhizomes (1-3 mm in diam.) ( Fig. 7; S. X. Liu et al. 2016018). The herbarium specimens from Wushan (B. L. Guo & X. Z. Luo 89003) and Wuxi (B. L. Guo A15) also presented creeping rhizome. The rhizome was therefore revised as long creeping or compact.
The leaves of some individuals were densely covered with golden-yellow villi on the abaxial side. And the petiolule, petiole and flowering stem were covered with multicellular glandular-hairs and golden-yellow villous, which are densest at the nodes. However, these indumentum characters were not stable and exhibited great variation in colour and thickness. Depending on the individuals and/or developmental stage (young or old), the indumentum of the abaxial villi varied continuously from dense to sparse, and the colour was also not always typical golden-yellow, ranging from white to yellow.
Our field investigation showed that the flowering stem primarily had 2 opposite trifoliolate leaves. In addition, abundant variations, such as 1 unifoliolate or trifoliolate leaf, 2 opposite unifoliolate leaves or 2 alternate leaves with one trifoliolate and the other unifoliolate, were also observed. And all these styles were presented in two specimens (Wushan: B. L. Guo  Key to the species of Epimedium in China In total, 57 species and 6 varieties have been described from China, although 16 of these were designated as synonyms. Epimedium platypetalum var. tenue B.L.Guo & P.K.Hsiao was treated as synonym of E. pauciflorum (Stearn 2002). Epimedium simplicifolium T.S.Ying and E. chlorandrum Stearn were treated as synonyms of E. acuminatum (Zhang et al. 2011;Zhang et al. 2015). Epimedium sagittatum var. guizhouense S.Z.He & B.L.Guo and E. pudingense S.Z.He, Y.Y.Wang & B.L.Guo were treated as a synonym of E. sagittatum (Sieb. &Zucc.) Maxim. and E. sagittatum var. glabratum T.S.Ying, respectively (Xu et al. 2014b (Zhang et al. 2015). Epimedium lishihchenii Stearn and E. baojingense Q.L.Chen & B.M.Yang were treated as subspecies and variety of E. franchetii Stearn, respectively (Liu et al. 2016). Additionally, Epimedium tianmenshanense T.Deng, D.G.Zhang & H.Sun is an insufficiently known taxon, due to the petals being found to be highly variable in morphology (both shape and size) (our field observation). Therefore, 45 species, 1 subspecies (E. franchetii ssp. lishihchenii) and 2 varieties (E. sagittatum var. glabratum T.S.Ying, E. franchetii var. baojingense) are recognised.

Discussion
Although the genus Epimedium is colourful in flower, from white, through yellow, to rose and purple, intraspecific flower colour variation is relatively uncommon. Before this study, only the polymorphism of E. grandiflorum and E. acuminatum has been described in the monograph or in the Flora of China (Stearn 2002;Ying et al. 2011). In addition, the colour variation of E. glandulosopilosum (yellow or pale purple) (Zhang et al. 2011) and E. pseudowushanense B.L.Guo (pale purple rose or purple, occasionally pale yellow) (Guo et al. 2007) have been mentioned. Since 2012, systematic studies and illustrations on morphology variation of Epimedium have been conducted by our group and we found five species with abundant intraspecific variations in flower colour. Epimedium acuminatum was the species with the best-known colour variation. The flower colour of E. acuminatum was described as "white, yellow, rose-purple or pale violet" in Flora of China (Ying et al. 2011). And variations ranging from whitish, pale yellow, yellow, pale purple, purple-whitish to purple or reddish-purple have been noted in the specimens. Additionally, two species, E. simplicifolium and E. chlorandrum, akin to E. acuminatum have been described. The flower colour of E. simplicifolium was originally described as yellow (Ying 1975). It was subsequently revised to be purple or yellow (Ying 2001), and then revised again to reddish purple by the same author (Ying et al. 2011). Epimedium chlorandrum is notable for its anthers being green and the flowers being very pale primrose yellow (Stearn 1997). But yellow flower and polymorphism from yellow to pale reddish-purple have been described in its specimens B. L. Guo 0606 and B. L. Guo 0608, respectively. In 2015, Zhang et al. found that the diagnostic characters of E. simplicifolium (with unifoliolate leaves) and E. chlorandrum (with green anthers and pollen) were within the range of morphological variations of E. acuminatum and finally were treated as synonyms of E. acuminatum (Zhang et al. 2011(Zhang et al. , 2015. Now, all of these descriptions and revisions can be well explained. Epimedium acuminatum is one of the most widespread species in the genus, and exhibits much variation in flower colour and other morphological characters (Zhang et al. 2015). And the polymorphism of flower colour exists both among populations and among individuals within populations.
It is very interesting and unexpected that we observed abundant intraspecific flower colour diversity. Our study suggests that the flower colour is not as stable as previously imagined. Therefore, flower colour seems to be a feature liable to great variation within some species and its taxonomic value should be discounted. From the geographical distribution of the five species, the results are consistent with the viewpoint that the comparatively unstable species often occur in western China where the genus is best represented and its evolution is still ongoing (Stearn 2002).
Although abundant flower colour variation has been observed in the present study, the reason for, or mechanism of, the colour variation is still unclear. The natural variation in flower colour may occur via the deposition of various anthocyanin pigments (Yang et al. 2010). Substantial variation in flower colour can be due to the differences in the presence, amount, or type of the carotenoid pigments (Wessinger 2015). In general, flower colour polymorphisms appeared to be a natural starting point. Flower colour polymorphisms are widely used as model traits from genetics to ecology (Alan 2007;Takahashi et al. 2013;Wang et al. 2017;Vaidya et al. 2018). In the present study, geographic variation in flower colour pattern within E. acuminatum showed a north-south geographic trend. The specimens with yellow flowers are mainly from the northern region of its distribution, while those with purple flowers are usually from the southern. The specimens with mixed colours occurred in the northwest of its distribution area (Fig. 2). The geographic pattern suggests that the variation in colour may be influenced by climate and ecology. Additionally, variation in flower colour has commonly been interpreted as adaptive. The differentiation in flower colour therefore was considered an important factor in promoting plant speciation (Bradshaw et al. 1995;Matsumura et al. 2006;Hopkins and Rausher 2011) or promoting adaptive, resulting from the disruptive selection by different pollinators (Mascó et al. 2004;Irwin and Strauss 2005;Veiga et al. 2015). On the other hand, the reproductive system may influence the patterns of variation in some taxa, and might account for the morphological complexity. Some colours, especially the continuous colour variation or the transition colours, may be generated by hybridisation, including hybrid speciation, historical hybridisation and ongoing speciation. Strong evidence for an outbreeding system and no internal barrier to hybridisation (high incompatibility and cross-ability) in Epimedium species has been proved (Suzuki 1983(Suzuki , 1984Sheng et al. 2011). Epimedium are promiscuous, with bees creating garden hybrids and natural hybrids in the wild (Stearn 2002;Avent 2010;Horie et al. 2012). So far, more than 20 hybrids have been found in field or in garden cultivation (Stearn 2002;Avent 2010). For example, in the wild, on Emei Mountains, E. acuminatum was hybridised with E. fangii and produced a hybrid swarm (named E. × omeiense) (Stearn 1995). Hybrids tend to have different colours which enriches the colour variation (Stearn 2002;Avent 2010;Horie et al. 2012). Hybridisation may be one of the mechanisms of colour variation in Epimedium.
From another perspective, our results could have important implications for the utilisation of germplasm. Bearing lovely foliage and graceful flowers, Epimedium plants were previously mainly introduced as garden plants in Europe and America (Stearn 2002;Ward 2004). The species from China and their hybrids are about to set the gardening world on fire (Probst 1998). With great commercial prospects, Epimedium had received increased attention from cultivators. The abundant flower colour variations of intraspecific are of great significance in promoting their ornamental value and in creating many possibilities (Avent 2010).

Conclusion
In this study, based on the extensive field investigation of populations during flowering seasons, comprehensive descriptions and illustrations for five Epimedium species were established. The flower colour used to be an important character in delimiting species in Epimedium (Stearn, 2002;Ying et al. 2011), but according to our results, flower colour might be too variable in the genus to be used in species delimitation. The flower colour and other characteristic variations in Epimedium are more extensive and complex than previously recognised. Therefore, it is not surprising that the Epimedium is taxonomically difficult and bewildering. The present study suggests that the flower colour is not constant at least in some species of Epimedium, which could be important to further taxonomic and evolutionary study.