A taxonomic revision of three Chinese spurless species of genus Epimedium L. (Berberidaceae)

Abstract Due to some common or similar features (e.g., small leaf, spurless, yellow flower), three Chinese species of the genus Epimedium (Berberidaceae), E. ecalcaratum, E. platypetalum, and E. campanulatum, are controversial based on morphological characteristics. In the present study, the descriptions of morphological characteristics for the three species were revised based on extensive studies and observations both in field and in herbaria. In general, E. ecalcaratum has long creeping rhizomes 1–3 mm in diameter, two alternate or opposite trifoliolate leaves, 7–14 flowers, and petals obovate and apex subacute. Epimedium platypetalum has short or long-creeping rhizomes 1–3 mm in diameter, one trifoliolate leaf, 2–6 flowers, and petals oblong and apex rounded. Epimedium campanulatum has compact rhizomes 4–6 mm in diameter, two alternate or opposite trifoliolate leaves, 15–43 flowers, and petals obovate and apex rounded. Through comparison, we found that despite the close affinity of these three species, they can be distinguished by rhizome differences, stem-leaves, the morphology of flower (e.g., petals), and the number of per inflorenscence.


Introduction
As a traditional Chinese herbal medicine, Epimedium has been widely used for "strengthening the kidney" and "reinforcing bone" for thousands of years (Jiang et al. 2016). Forty-nine species have been reported in China (Ogisu and Rix 2011, Xu et al. 2014, Zhang et al. 2014, Zhang et al. 2015, Zhang et al. 2016, Wei et al. 2017. Stearn (2002) divided the Chinese Epimedium into four series: Campanulatae, Davidianae, Dolichocerae, and Brachycerae. For a long time, E. ecalcaratum G. Y. Zhong, E. platypetalum K. I. Meyer, E. campanulatum Ogisu and E. shuichengense S. Z. He were recognized in the series Campanulatae (Stearn 2002). Among them, E. shuichengense was once thought to be a very special species with spurless petals, representing a transition stage of floral evolution from series Campanulatae (spurless small-flowered species) to series Davidianae (long-spurred species) (Stearn 2002). However, according to field investigations in the type locality, Zhang et al. (2015a) confirmed that E. shuichengense belongs to series Davidianae while E. reticulatum C. Y. Wu ex S. Y. Bao truly belongs to series Campanulatae. Therefore, four species, namely, E. reticulatum, E. ecalcaratum, E. platypetalum, and E. campanulatum were included in the series Campanulatae in China.
However, E. reticulatum is distinctive and can be easily distinguished from other spurless species. The petals of E. reticulatum are flat with a slightly cucullate base, the flower size is obviously smaller (about 7 mm) than other spurless species (about 10 mm), and its leaflets are thickly leathery with conspicuous reticulate veins on both sides (Bao 1987;Zhang et al. 2015a). Therefore, the present study focuses on the remaining three species in series Companulatae in China that can easily be confused.
On the other hand, numerous Epimedium species have been described without extensive morphological observation. There is also a lack of both field investigations and other studies. Epimedium ecalcaratum was described as having compact rhizomes based on very limited samples (Zhong 1991), yet this character was adopted by the Flora Reipublicae Popularis Sinicae (Ying 2001). Since E. platypetalum was described in 1922, there has been very little research concerning morphological observations for E. platypetalum. According to the description based on several individuals grown at Blackthorn Nursery Kilmeston, E. campanulatum is morphologically similar to E. ecalcaratum and E. platypetalum (Ogisu 1996) and it was later treated as a insufficiently known species by Ying et al. (2011). In general, very little is known about the range of variation of characters, variation patterns, and the taxonomic value of these allied species.
Based on extensive studies of the three spurless Epimedium species, both in field investigations (during flowering seasons) and in herbaria, the aim of this study was to 1) revise and complete morphological descriptions, and 2) compare the morphological differences among the three similar species.

Field investigation
Field investigations on the germplasm resource and morphological observations have been conducted from 2012 to 2016. Field work was done in Hubei, Shanxi, Chongqing and Sichuan Province, China. A total of 120 individuals (30 individuals per population) from four populations of three spurless species, E. ecalcaratum (two populations), E. platypetalum and E. campanulatum, were collected from Sichuan and Shanxi Provinces (Table 1). All populations were investigated and collected during the flowering, as the floral properties are significant for the taxonomy of Epimedium species. To capture variation within populations, 30 individuals per population were observed and sampled. Quantitative measurements on rhizome diameter, height of flowering stem, length of inflorescence, number of flowers, and length and width of the middle leaflet were recorded for each individual. The average data were processed using SPSS 19.0 software. Concurrently, the folloeing discrete morphological characters were observed: the rhizome; pedicel, petiole, underside of leaflet hair characteristics; shape and number of leaflets; number of stem-leaves; inflorescence; leaflets and flowers; shape and color of inner sepals; and shape of petals.

Specimen examination
All 120 individuals of the three species were transplanted at the Jiangxi University of Traditional Chinese Medicine, China. Herbarium specimens were examined from the following herbaria: Chinese Academy of Medical Sciences, Peking Union Medical College Institute of Medicinal Plant Development (IMD); Institute of Botany, Chinese Academy of Sciences (PE); Chongqing Academy of Chinese Materia Medica (SM); Virtual Museum System (HX); Nanjing University (N); and Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (NAS).

Geographical distribution
Based on field investigations and herbarium specimens, E. ecalcaratum, E. platypetalum and E. campanulatum were stenochoric species (Fig. 1). Epimedium platypetalum has previously been collected in Sichuan; however, it was not observed during our field investigations, likely due to habitat destruction. Therefore, the populations that can be collected were very limited.

Quantitative characters analysis
Quantitative morphological data from E. ecalcaratum, E. platypetalum, and E. campanulatum are presented in Table 2. Among the three close allies, E. campanulatum was easily identified by its long flowering stem and inflorescence, the largest number of flowers, and the stoutest rhizome. Epimedium ecalcaratum and E. platypetalum were very similar in terms of quantitative traits, but E. platypetalum has fewer flowers (2-8 flowers per individual; population mean of four flowers per individual). The main quantitative characters of the two populations of E. ecalcaratum were similar, only the length of the flowering stem was found having a slight difference.

Discrete morphological characters
The main discrete morphological characters of E. ecalcaratum, E. platypetalum and E. campanulatum are presented in Table 3. The three species all had glandular hairy inflorescence and pedicels; flat, spurless, yellow, pendulous flowers; and obovate petals. Although having much in common with its close allies, E. campanulatum differed by having compound inflorescences and cup-shaped flowers, while E. ecalcaratum differed because of the slightly saccate base of the petals, creating a slightly shouldered flower base (Fig. 2). Among the three species, the most diverse characters are the number of leaflets, the number of stem-leaves, and the arrangement of leaves on the stem (Table 3). The rhizome also presented a clear differentiation among the three species.

Discussion
Key to species of ser. Campanulatae The protologue (Zhong 1991) and the subsequent description in Flora of China (Ying 2001) both described the compact rhizome of E. ecalcaratum. We re-examined the holotype and conducted fieldwork in its type locality. However, individuals of E. ecalcaratum in the field all had long creeping rhizomes, slender nodes with numerous fibrous roots, 1-3 mm in diameter, and internodes sometimes to 30 cm. In the genus Epimedium, this situation may not be rare. The form of the rhizome, specifically the degree of elongation and thickness, and also the average size of the terminal winter-bud, is constant for each species, and sometimes offers contrasts of taxonomic value (Stearn 2002). But Stearn (1997) pointed out that the different rhizome forms among some Epimedium species can sometimes be very evident, sometimes more subtle. For example, the major difference between E. leptorrhizum Stearn and E. brachyrrhizum Stearn was that the former had a very slender elongated rhizome while the latter bore a more compact clump-forming rhizome. However, examination of a series of E. leptorrhizum specimens showed that its rhizome was often slender and long-creeping but occasionally thicker and compact (Zhang et al. 2015b). In addition, the protologue of E. lishihchenii Stearn differs from E. franchetii Stearn was in having a long-creeping rhizome (Stearn 1997). However, our field observation based on a population found that E. lishihchenii 20% of individuals had compact rhizomes (Liu et al. 2016).
Due to the slender elongated rhizome and small broadly ovate or almost orbicular leaflets, Guo et al. (1993) described a variety, E. platypetalum var. tenuis B. L. Guo et P. G. Hsiao. Stearn (1995) assessed E. platypetalum var. tenuis and found it differed from E. platypetalum bacause of its long-spurred flowers. Then, he treated E. platypetalum var. tenuis as a synonym of E. pauciflorum K. C. Yen, a new species that was published after the description of E. platypetalum var. tenuis (Yen 1994;Stearn 1995). In a more recent study, Ying et al. (2011) still treated E. platypetalum var. tenuis as a synonym of E. platypetalum. We re-examined the specimens of E. platypetalum var. tenuis, and found the taxon had obviously long-spurred petals (1.7 cm) and long inner sepals (1.4 cm) that were completely different with E. platypetalum. In addition, E. platypetalum var. tenuis has sympatric distribution with E. pauciflorum. Therefore, we agree with Stearn (1995) that E. platypetalum var. tenuis should be revised as a synonym of E. pauciflorum.
The number of stem-leaves was believed to be stable within a species and important for taxonomy, and three informal groups have been divided by the normal number of stem-leaves (Stearn 2002). Stem-leaves, however, are not so unvarying as initially supposed. Stearn (2002), Zhang et al. (2015), and our field observation have recognized and recorded some variation on the number of leaves. For example, usually two opposite or occasionally three whorled leaves were observed in E. sagittatum (Sieb. et Zucc.) Maxim., E. acuminatum Franch., E. myrianthum Stearn and E. franchetii, and one leaf or two leaves in E. epsteinii Stearn, E. flavum Stearn, E. leptorrhizum and E. pauciflorum. It is significant that the comparatively unstable species occur in western China, where the genus is best represented and where its evolution may still be proceeding (Stearn 2002). Population is the basic unit of evolution (Chen 2016) and the most important unit to study the formation of species (Chen and Wang 1986;Nooteboom 1992;Hong 2016). Thus, morphological differences recorded among individuals in a population should not be ignored and may be more obvious than populations in sometimes (Yang 1991;Jonas et al. 1999). Previous studies on the taxonomy of the genus Epimedium were almost always based on limited samples, or several individuals cultivated abroad (Ying 2001). Our investigations based on populations in their native habitat found that the number of leaves and the habit of the flowering stem presented abundant variation (Fig. 3).
The protologue for E. ecalcaratum described that its stem-leaves are usually opposite with two trifoliolate leaves, occasionally alternate with two trifoliolate leaves or three trifoliolate leaves (Zhong 1991). Our investigation showed that it usually had two alternate or opposite trifoliolate leaves, sometimes three alternate trifoliolate leaves, occasionally two opposite 5-foliolate leaves, or rarely three or four whorled unifoliolate, trifoliolate and/or 5-foliolate leaves (Fig. 3A-G). In the protologue of E. campanulatum (Ogisu 1996), the species was described with one leaf or two usually alternate, rarely opposite leaves, but our investigation showed that it usually had two alternate (mostly two trifoliolate, and occasionally one trifoliolate and one simple) or opposite leaves, sometimes three alternate leaves, and one trifoliolate leaf was also occasionally observed (Fig. 3M-Q). Our study clearly shows that the number and insertion of the leaves and the number of leaflets varies in this species. Upon extensive specimen examination, Zhang et al. (2011Zhang et al. ( , 2015b observed that the leaves of E. simplicifolium T. S. Ying were mainly unifoliolate, occasionally trifoliolate, and the leaves of E. acuminatum Franch. may be unifoliolate. Subsequently, E. simplicifolium was synonymized with E. acuminatum (Zhang et al. 2011). Hence, as a taxonomist, it is important to study as many collections as possible (Xu 1998).
Although having much in common with E. platypetalum and E. campanulatum, E. ecalcaratum differs in having slightly a saccate petal base, creating a slightly shouldered base to the flower (Stearn 2002), which is in agreement with our field observations ( fig. 2A, B, D). According to Stearn's research, the character could be regarded as moving towards development of a nectar-producing spur, and he published a photo to show the petals are typically without a spur, but may have varying degrees of small spurs (Stearn 2002: 53, fig. 18). This may indicate that E. ecalcaratum represented a transitional stage in floral evolution from series Campanulatae (spurless) to series Davidiance (spur with basal lamina). Spur variations have not been observed in the present study, and are not supported by specimens or other literature.

Conclusions
Despite similarity in leaf size and flat, suprless, yellow flowers, E. ecalcaratum, E. platypetalum and E. campanulatum could be distinguished by the following characters: rhizome form, number of stem-leaves, leaflets, flowers, inflorescence, and petals and inner sepal shape (Table 4 and Fig. 2).