# The Cytology, Isozyme, HPLC Fingerprint, and Interspecific Hybridization Studies of GenusEpimedium (Berberidaceae)

Authors: Lin-Jiao Wang; Mao-Yin Sheng
Journal: ISRN Pharmacology
 (2013)

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## Abstract

104 samples from 27 accessions belonging to 12 species of genusEpimedium were studied on the basis of cytology observation, POD (i.e., peroxide) isozyme, high performance liquid chromatography (i.e., HPLC) fingerprint, and interspecific hybridization. The cytology observation showed karyotypes of twelve species studied; all are 2A symmetry type of Stebbins standard and similar to each other, and except for karyotype of E. leptorrhizum which is 2n = 2x = 8m (2SAT) + 4sm, the rest  are 2n = 2x = 6m (2SAT) + 6sm. Chromosomes C-banding of barrenwort species varies, with 15 to 22 bands, consisting of centromeric bands, intercalary bands, terminal bands, and middle satellite bands. Results of POD isozyme showed that the zymographs vary greatly and sixteen bands were detected in the eleven species, and each species has its own characteristic bands different from the others. Studies on the HPLC fingerprint showed that the HPLC fingerprint of different species has characteristic peaks, divided into two regions (retention time < 10 min and retention time > 10 min). Results of interspecific hybridization showed that crosses of any combination among seven species studied are successful and the rates of grain set vary greatly. Based on these results, the system and phylogeny of this genus were inferred.


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## Body

## 1. Introduction

Barrenwort (Epimedium, Berberidaceae) is a traditional Chinese medicine named as Yinyanghuo, Xianlinpi, and Yangheye in China [1–3]. The plant is effective in strengthening kidneys and curing rheumatism, widely used in the treatment of osteoporosis, hypertension, coronary heart disease [4], caducity, cancer, and so on [1–3]. Many flavonol glycosides have been isolated from a number of Epimedium plants, though the relation between these flavonol glycosides and the pharmacological activities of the drug is still obscure [4]. In Japan, Europe, and America, barrenwort are also very popular as graceful garden plants.Epimedium is a genus of the Old World. Members disperse from Japan to Algeria and mainly occur in eastern Asia and the Mediterranean lands [5]. Approximately 80% of the total species are found in central-southeastern China [6]. Linnaeus recorded this genus and its type species E. alpinumin 1753. After that, Morren and Decaisne (1834); Franchet (1886); Komarov (1908); and Stearn (1938, 2002) made monographic and systematic study of Epimedium, respectively. Up till now, more than 60 species of Epimedium are recognized. Stearn [5, 7] established the most comprehensive classification system of this genus. In his monograph, he arranged the genus into two subgenera,Epimedium L. and Rhizophyllum (Fischer & Meyer) Stearn, mainly based on whether the flowering stem has leaves or not, and he divided the subgenus, Epimedium, into four sections mainly according to their geographical distribution and infrageneric relationship. Among the four sections, members of section Diphyllon (Kom.) Stearn were all endemic to China and subdivided into four series: Campanulatae Stearn, Davidianae Stearn, Dolichocerae Stearn, and Brachycerae Stearn based on corolla characteristics such as petal type, the form and relative size of the inner sepals and petals, and flower dimension.Up to now, lots of works have been adopted to deduce the system and evolution of this genus such as pollen morphology [8, 9], cytology [10, 11], isozyme [12], molecular biology [13–16], biogeography [6, 17], chemical classification [4, 12, 18], and so on. Results showed that the infrageneric classification was generally consistent with its phytogeographic distribution, and the phylogenetic relationship among sections was basically clear in Epimedium. However, for lots of Epimedium species with multifarious petals are nested together in China and the petal evolved is quite controversial, the system of Chinese species is still not revealed well [18]. Recently, many further researches were performed for the system and phylogeny of Epimedium[17, 19, 20]. As an intractable genus in taxonomy, however, there still remain several problems about the classification and phylogenetic relationship of Epimedium species, especially for the Chinese Epimedium species. As a result, it is essential to establish a natural arrangement of Epimedium.In this paper, 104 samples from 27 accessions belonging to twelve barrenwort species were systemically studied on the cytology, peroxide (POD) isozyme, high performance liquid chromatography (HPLC) fingerprint, and interspecific hybridization to infer the system and phylogeny of this genus.


## 2. Materials and Methods

### 2.1. Plant Materials

We collected 104 samples from 27 accessions belonging to twelve barrenwort species (Table1) and planted them in pots filled with compost (one plant per pot). All the pots were kept in greenhouses at the Institute of Plant Genetics and Breeding (IPGB) under identical conditions. The entire study was carried out at the same institute.Table 1
Epimedium accessions used in this study.
Accessions
Species
Native to
Symbols
Speciman no.
Size of flower
EP04100701AC
E. acuminatum Franch
Guizhou, Guiyang, China
AC1
04100701
Large
EP05050709AC
E. acuminatum Franch
Guizhou, Yanhe, China
AC2
05050709
Large
EP05101001AC
E. acuminatum Franch
Guizhou, Mentan, China
AC3
05101001
Large
EP05101002AC
E. acuminatum Franch
Yunnan, Kunming, China
AC4
05101002
Large
EP05050801AC
E. acuminatum Franch
Guizhou, Dejiang, China
AC5
05050801
Large
EP05021002YI
E. yinjiangense M. Y. Sheng and Q. F. Chen
Guizhou, Yinjiang, China
YI1
05021002
Large
EP05021003YI
E. yinjiangense M. Y. Sheng and Q. F. Chen
Guizhou, Yinjiang, China
YI2
05021003
Large
EP05050403LU
E. luodianense M. Y. Sheng and Q. F. Chen
Guizhou, Luodian, China
LU1
05050403
Large
EP04120001LU
E. luodianense M. Y. Sheng and Q. F. Chen
Guizhou, Pingtan, China
LU2
04200001
Large
EP04081205SI
E. simplicifolium T. S. Ying
Guizhou, Yanhe, China
SI1
04081205
Large
EP04081208SI
E. simplicifolium T. S. Ying
Guizhou, Yanhe, China
SI2
04081208
Large
EP06012301FR
E. franchetii Stearn
Guizhou, Tongren, China
FR
06012301
Large
EP04041001WU
E. wushanense T. S. Ying
Guizhou, Kaili, China
WU1
04041001
Large
EP05051102WU
E. wushanense T. S. Ying
Guizhou vivariuam, China
WU2
05051102
Large
EP05032101WU
E. wushanense T. S. Ying
Guizhou, Guiyang, China
WU3
05032101
Large
EP04061505LE
E. leptorrhizum Stearn
Guizhou, Dejiang, China
LE1
04061505
Large
EP04091201LE
E. leptorrhizum Stearn
Guizhou, Wuchuan, China
LE2
04091201
Large
EP04061501LE
E. leptorrhizum Stearn
Guizhou, Yinjiang. China
LE3
04061501
Large
EP04052201LE
E. leptorrhizum Stearn
Guizhou, Guiyang, China
LE4
04052201
Large
EP05100801DA
E. davidii Franch
Yunnan, Kunming, China
DA
05101001
Large
EP04072202AL
E. alpinum L.
Munich, German
AL1
04072201
Large
EP04072201AL
E. alpinum L.
Munich vivariuam, German
AL2
04072201
Large
EP05092302PU
E. pubigerum (DC.) Morren and Decne.
Munich, German
PU1
05092302
Large
EP05092301PU
E. pubigerum (DC.) Morren and Decne.
Munich vivariuam, German
PU2
05092301
Large
EP03100202SA
E. sagittatum (Sieb. et. Zucc.) Maxim.
Guizhou vivarium, China
SA1
03100202
Small
EP03100201SA
E. sagittatum (Sieb. et. Zucc.) Maxim.
Guizhou Shibing, China
SA2
03100201
Small
EP04050401MY
E. myrianthum Stearn
Guizhou, Shibing, China
MY
04050401
Small


### 2.2. Karyotype Analyses

Somatic chromosomes were studied in root meristems of individuals which were pretreated inα-bromine naphthalin at 23°C for 5 h, then fixed in ethanol : acetic acid (3 : 1) for 4~12 h, and stored in 70% aqueous ethanol. Root tips were stained according to Feulgen technique, meristems were macerated in 1 mol/L hydrochloric acid at 60°C for 10 minutes before squashing, and slides were made permanent using Balata as the mounting medium. At least 10 metaphases were drawn for each population using an Olympus BX51 camera (Olympus, Japan), selecting the five best for measurements. The nomenclature used for the description of the chromosome morphology is that proposed by Levan et al. [21].For the numerical characterization of the karyotypes, the following parameters were calculated: (1) total chromosome length of the haploid complement (TCL); (2) mean chromosome length (CL); (3) mean centromere index (CI); (4) intrachromosomal asymmetry index(A1)=1-[∑(b/B)/n]; and (5) interchromosomal asymmetry index (A2)=s/x, where b and B are the mean length of short and long arms of each pair of homologues, respectively, n is the number of homologues, s is the standard deviation, and x is the mean chromosome length. Karyotype asymmetry has been determined using the categories of Stebbins [22].


### 2.3. Giemsa C-Band Analyses

Somatic chromosomes were studied in root meristems of individuals which were pretreated in 0.2% aqueous colchicine at 23°C for 4 h, then fixed in ethanol : acetic acid (3 : 1) for 24 h, and stored in 70% aqueous ethanol; meristems were squashed in 45% acetum. Cover glasses were uncovered after freezing, and then samples were set in air at 23°C for five days before being treated by Giemsa chromosome banding. The improved BSG method of chromosome C-banding was used. Mature samples were treated in 0.2 mol/L hydrochloric acid for 50 minutes, treated in 50% aqueous Ba(OH)2 at 60°C for 30 minutes after being washed by water, then treated in 2 × SSC solution at 60°C for 5 h after being washed by water, and finally dyed in 3% aqueous Giemsa (pH 6.8) for 1 h. Chromosomes C-bandings among species were compared by the method of Li [23].


### 2.4. PAGE Analyses of POD Isozyme

The polyacrylamide gel electrophoresis (PAGE) method of POD isozyme analyses was described by Hu and Wang [24]. The POD was extracted from tender, 2-week-old leaves. The parameters of bio-gel for separation were as follows: T (total concentration of Acr and Bis) = 7.5% and C (the rate of B is in T) = 2.6%. Rf value = moving length of the band/moving length of the front line. The coefficient (c) and dissimilarity coefficient (d) of zymogram were used to analyse the relation among species. c=(2w/(a+b))×100%, where a= the number of zymogram bands of species A, b= the number of zymogram bands of species B, and w= the number of zymogram bands common to species A and B, and d=100%-c. Idiograms were drawn based on the results and analyzed.


### 2.5. HPLC Fingerprint of Flavonoids

The HPLC fingerprints were estimated by following the method described by CCP [25]. The HPLC system consisted of an LC2010 and CLASS-VP Ver6.12 computer system (all components from Shimadzu, Kyoto, Japan). A reversed-phase C18 (YMC, Japan) column (150 × 4.6 mm) was used for separation. The mobile phase consisted of acetonitrile and water (25 : 75). The flow rate was kept constant at 1.0 mL/min at room temperature (30°C). The detection was performed at 270 nm using an SPD-M10A VP photodiode array detector. The samples delivered into the system, one at a time, measured 10 μL each. All the samples used in the study were from mature leaves.


### 2.6. Interspecific Hybridization

Seven species,E. myrianthum, E. acuminatum, E. wushanense, E. letorrhizum, E. luodianense, E. simplicifolium,andE. yinjiangense grown in green house, were selected as female and male parents, crossed with each other. As flowers opened (before anther dehiscence), the species chosen as female parents were emasculated and pollinated by rubbing them with newly dehisced anthers of the male parents. More than 200 flowers were pollinated per cross. Remaining buds in the inflorescences were removed. Twenty days later, the rate of grain set (%) = (the number of grain set/the number of pollinated flowers) × 100. Based on the rate of grain set, the relations of these seven species were compared by the hierarchical cluster of SPSS [26].


## 2.1. Plant Materials

We collected 104 samples from 27 accessions belonging to twelve barrenwort species (Table1) and planted them in pots filled with compost (one plant per pot). All the pots were kept in greenhouses at the Institute of Plant Genetics and Breeding (IPGB) under identical conditions. The entire study was carried out at the same institute.Table 1
Epimedium accessions used in this study.
Accessions
Species
Native to
Symbols
Speciman no.
Size of flower
EP04100701AC
E. acuminatum Franch
Guizhou, Guiyang, China
AC1
04100701
Large
EP05050709AC
E. acuminatum Franch
Guizhou, Yanhe, China
AC2
05050709
Large
EP05101001AC
E. acuminatum Franch
Guizhou, Mentan, China
AC3
05101001
Large
EP05101002AC
E. acuminatum Franch
Yunnan, Kunming, China
AC4
05101002
Large
EP05050801AC
E. acuminatum Franch
Guizhou, Dejiang, China
AC5
05050801
Large
EP05021002YI
E. yinjiangense M. Y. Sheng and Q. F. Chen
Guizhou, Yinjiang, China
YI1
05021002
Large
EP05021003YI
E. yinjiangense M. Y. Sheng and Q. F. Chen
Guizhou, Yinjiang, China
YI2
05021003
Large
EP05050403LU
E. luodianense M. Y. Sheng and Q. F. Chen
Guizhou, Luodian, China
LU1
05050403
Large
EP04120001LU
E. luodianense M. Y. Sheng and Q. F. Chen
Guizhou, Pingtan, China
LU2
04200001
Large
EP04081205SI
E. simplicifolium T. S. Ying
Guizhou, Yanhe, China
SI1
04081205
Large
EP04081208SI
E. simplicifolium T. S. Ying
Guizhou, Yanhe, China
SI2
04081208
Large
EP06012301FR
E. franchetii Stearn
Guizhou, Tongren, China
FR
06012301
Large
EP04041001WU
E. wushanense T. S. Ying
Guizhou, Kaili, China
WU1
04041001
Large
EP05051102WU
E. wushanense T. S. Ying
Guizhou vivariuam, China
WU2
05051102
Large
EP05032101WU
E. wushanense T. S. Ying
Guizhou, Guiyang, China
WU3
05032101
Large
EP04061505LE
E. leptorrhizum Stearn
Guizhou, Dejiang, China
LE1
04061505
Large
EP04091201LE
E. leptorrhizum Stearn
Guizhou, Wuchuan, China
LE2
04091201
Large
EP04061501LE
E. leptorrhizum Stearn
Guizhou, Yinjiang. China
LE3
04061501
Large
EP04052201LE
E. leptorrhizum Stearn
Guizhou, Guiyang, China
LE4
04052201
Large
EP05100801DA
E. davidii Franch
Yunnan, Kunming, China
DA
05101001
Large
EP04072202AL
E. alpinum L.
Munich, German
AL1
04072201
Large
EP04072201AL
E. alpinum L.
Munich vivariuam, German
AL2
04072201
Large
EP05092302PU
E. pubigerum (DC.) Morren and Decne.
Munich, German
PU1
05092302
Large
EP05092301PU
E. pubigerum (DC.) Morren and Decne.
Munich vivariuam, German
PU2
05092301
Large
EP03100202SA
E. sagittatum (Sieb. et. Zucc.) Maxim.
Guizhou vivarium, China
SA1
03100202
Small
EP03100201SA
E. sagittatum (Sieb. et. Zucc.) Maxim.
Guizhou Shibing, China
SA2
03100201
Small
EP04050401MY
E. myrianthum Stearn
Guizhou, Shibing, China
MY
04050401
Small


## 2.2. Karyotype Analyses

Somatic chromosomes were studied in root meristems of individuals which were pretreated inα-bromine naphthalin at 23°C for 5 h, then fixed in ethanol : acetic acid (3 : 1) for 4~12 h, and stored in 70% aqueous ethanol. Root tips were stained according to Feulgen technique, meristems were macerated in 1 mol/L hydrochloric acid at 60°C for 10 minutes before squashing, and slides were made permanent using Balata as the mounting medium. At least 10 metaphases were drawn for each population using an Olympus BX51 camera (Olympus, Japan), selecting the five best for measurements. The nomenclature used for the description of the chromosome morphology is that proposed by Levan et al. [21].For the numerical characterization of the karyotypes, the following parameters were calculated: (1) total chromosome length of the haploid complement (TCL); (2) mean chromosome length (CL); (3) mean centromere index (CI); (4) intrachromosomal asymmetry index(A1)=1-[∑(b/B)/n]; and (5) interchromosomal asymmetry index (A2)=s/x, where b and B are the mean length of short and long arms of each pair of homologues, respectively, n is the number of homologues, s is the standard deviation, and x is the mean chromosome length. Karyotype asymmetry has been determined using the categories of Stebbins [22].


## 2.3. Giemsa C-Band Analyses

Somatic chromosomes were studied in root meristems of individuals which were pretreated in 0.2% aqueous colchicine at 23°C for 4 h, then fixed in ethanol : acetic acid (3 : 1) for 24 h, and stored in 70% aqueous ethanol; meristems were squashed in 45% acetum. Cover glasses were uncovered after freezing, and then samples were set in air at 23°C for five days before being treated by Giemsa chromosome banding. The improved BSG method of chromosome C-banding was used. Mature samples were treated in 0.2 mol/L hydrochloric acid for 50 minutes, treated in 50% aqueous Ba(OH)2 at 60°C for 30 minutes after being washed by water, then treated in 2 × SSC solution at 60°C for 5 h after being washed by water, and finally dyed in 3% aqueous Giemsa (pH 6.8) for 1 h. Chromosomes C-bandings among species were compared by the method of Li [23].


## 2.4. PAGE Analyses of POD Isozyme

The polyacrylamide gel electrophoresis (PAGE) method of POD isozyme analyses was described by Hu and Wang [24]. The POD was extracted from tender, 2-week-old leaves. The parameters of bio-gel for separation were as follows: T (total concentration of Acr and Bis) = 7.5% and C (the rate of B is in T) = 2.6%. Rf value = moving length of the band/moving length of the front line. The coefficient (c) and dissimilarity coefficient (d) of zymogram were used to analyse the relation among species. c=(2w/(a+b))×100%, where a= the number of zymogram bands of species A, b= the number of zymogram bands of species B, and w= the number of zymogram bands common to species A and B, and d=100%-c. Idiograms were drawn based on the results and analyzed.


## 2.5. HPLC Fingerprint of Flavonoids

The HPLC fingerprints were estimated by following the method described by CCP [25]. The HPLC system consisted of an LC2010 and CLASS-VP Ver6.12 computer system (all components from Shimadzu, Kyoto, Japan). A reversed-phase C18 (YMC, Japan) column (150 × 4.6 mm) was used for separation. The mobile phase consisted of acetonitrile and water (25 : 75). The flow rate was kept constant at 1.0 mL/min at room temperature (30°C). The detection was performed at 270 nm using an SPD-M10A VP photodiode array detector. The samples delivered into the system, one at a time, measured 10 μL each. All the samples used in the study were from mature leaves.


## 2.6. Interspecific Hybridization

Seven species,E. myrianthum, E. acuminatum, E. wushanense, E. letorrhizum, E. luodianense, E. simplicifolium,andE. yinjiangense grown in green house, were selected as female and male parents, crossed with each other. As flowers opened (before anther dehiscence), the species chosen as female parents were emasculated and pollinated by rubbing them with newly dehisced anthers of the male parents. More than 200 flowers were pollinated per cross. Remaining buds in the inflorescences were removed. Twenty days later, the rate of grain set (%) = (the number of grain set/the number of pollinated flowers) × 100. Based on the rate of grain set, the relations of these seven species were compared by the hierarchical cluster of SPSS [26].


## 3. Results

### 3.1. Morphological Observation and Karyotype Analyses of Chromosomes

Based on the size of flower, twelve species can be grouped into two types, type one is small flower includingE. sagittatumand E. myrianthum,type two is large flower, the rest. The results of chromosomal observations of twelve species showed all species studied were diploid (2n=2x=12) and had one pair of middle satellite chromosomes. Expect for the middle satellite chromosomes of E. leptorrhizum, E. simplicifoliumand E. wushanense are in short arms of no. 2 chromosomes; the rest are no. 1 chromosomes. Tetraploid cells sporadically were found in E. letorrhizum root meristems among twelve species. The total length of haploid complement (TCL) of E. yinjiangenseand E. alpinum are obviously small, 29.23±2.36 μm and 32.96±1.34 μm, respectively. And the range of chromosome length of E. yinjiangense is 2.15~2.92 μm and E. alpinum 2.22~3.06 μm.The parameters (Table2), figures (Figure 1), and karyotypes (Figure 2) of twelve species metaphase chromosomes were studied. The results showed that their karyotypes are similar to each other, only including m and sm two types chromosome. Twelve species karyotypes are all 2A type of Stebbin’s asymmetry categories. Among them, the karyotype formulas of E. sagittatum, E. myrianthum, E. acuminatun, E. wushanense, E. simplicifolium, E. yinjianense, E. luodianense, E. davidii, E. pubigerum, E. franchetii, andE. alpinum are 2n=2x=6m (2SAT) + 6sm, and E. leptorrhizumis 2n=2x=8m (2SAT) + 4sm.Table 2
Karyotype formula (KF), satellite position (SAT), total length of the haploid complement (TCL), range of chromosome length (range), intrachromosome asymmetry index (A1), interchromosome asymmetry (A2), mean centromeric index (CI), and Stebbin’s asymmetry categories (ST) of the studied Epimedium species.
Species
KF
SAT
TCL ± SE
Range
A
1
A
2
CI ± SE
ST
E. acuminatum
4m + 6sm + 2m (SAT)
1s
45.23
±
0.78
3.08~4.77
0.38
0.17
37.54
±
0.15
2A
E. yinjiangense
4m + 6sm + 2m (SAT)*
1s
29.23
±
2.36
2.15~2.92
0.43
0.14
35.76
±
0.28
2A
E. luodianense
4m + 6sm + 2m (SAT)*
1s
47.88
±
1.24
2.28~4.87
0.38
0.17
36.69
±
0.31
2A
E. leptorrhizum
6m + 4sm + 2m (SAT)*
2s
46.00
±
1.04
3.06~4.44
0.34
0.14
39.00
±
0.14
2A
E. simplicifoilum
4m + 6sm + 2m (SAT)*
2s
45.43
±
2.01
3.05~4.17
0.42
0.11
36.06
±
0.52
2A
E. wushanense
4m + 6sm + 2m (SAT)*
2s
46.86
±
0.88
3.33~4.44
0.33
0.10
39.40
±
0.46
2A
E. alpinum
4m + 6sm + 2m (SAT)
1s
32.96
±
1.34
2.22~3.06
0.35
0.13
38.39
±
0.39
2A
E. davidii
4m + 6sm + 2m (SAT)*
1s
47.32
±
1.67
3.33~4.72
0.42
0.16
36.08
±
0.22
2A
E. pubigerum
4m + 6sm + 2m (SAT)
1s
38.59
±
0.96
2.50~3.61
0.32
0.14
39.88
±
0.18
2A
E. franchetii
4m + 6sm + 2m (SAT)*
1s
38.04
±
0.82
2.91~4.72
0.36
0.18
38.20
±
0.29
2A
E. myrianthum
4m + 6sm + 2m (SAT)*
1s
45.45
±
1.27
3.38~4.15
0.40
0.08
36.69
±
0.38
2A
E. sagittatum
4m + 6sm + 2m (SAT)
1s
41.88
±
1.01
3.27~4.68
0.34
0.18
38.69
±
0.13
2A
SE: standard error, m: metacentric, sm: submetacentric, st: subteleocentric, s: short arm, and *indicates first description of species.Figure 1
Somatic chromosome ofEpimedium, all species with 2n = 12. 1–10: large-flower group. 1: E. acuminatum. 2: E. yinjiangense. 3: E. luodianense. 4: E. leptorrhizum. 5: E. simplicifolium. 6: E. wushanense. 7: E. alpinum. 8: E. davidii. 9: E. pubigerum. 10: E. franchetii. 11-12: small-flower group. 11: E. myrianthum. 12: E. sagittatum. Scale bar = 2 μm.Figure 2
Karyotypes of twelve species metaphase chromosomes, all species with 2n = 12. 1–10: large-flower group. 1:E. acuminatum. 2: E. yinjiangense. 3: E. luodianense. 4: E. leptorrhizum. 5: E. simplicifolium. 6: E. wushanense. 7: E. alpinum. 8: E. davidii. 9: E. pubigerum. 10: E. franchetii. 11-12: small-flower group. 11: E. myrianthum. 12: E. sagittatum. Scale bar = 2 μm.


### 3.2. Giemsa C-Band Analyses of Chromosomes

Eight species,E. sagittatum, E. alpinum, E. pubigerum, E. acuminatum, E. simplicifolium, E. wushanense, E. letorrhizum, and E. davidii, were treated by chromosomes C-banding. The metaphase chromosomes of C-banding, Giemsa C-banding karyotypes and idiograms of C-banding karyotype can be seen in Figures 3, 4, and 5, respectively.Figure 3
The metaphase chromosomes C, banding in the eight species of genusEpimedium. 1: E. simplicifolium. 2: E. leptorrhizum. 3: E. wushanense. 4: E. alpinum. 5: E. davidii. 6: E. acuminatum. 7: E. pubigerum. 8: E. sagittatum. Scale bar = 2 μm.Figure 4
Giemsa C-banded karyotypes in the eight species of genusEpimedium. 1: E. simplicifolium. 2: E. leptorrhizum. 3: E. wushanense. 4: E. alpinum. 5: E. davidii. 6: E. acuminatum. 7: E. pubigerum. 8: E. sagittatum. Scale bar = 2 μm.Figure 5
Idiograms of C-banded karyotypes in the eight species of genusEpimedium. 1: E. simplicifolium. 2: E. leptorrhizum. 3: E. wushanense. 4: E. alpinum. 5: E. davidii. 6: E. acuminatum. 7: E. pubigerum. 8: E. sagittatum.Results showed that the chromosomes C-banding of eight species vary remarkably, with 15 to 22 bands, consisting of centromeric bands, intercalary bands, terminal bands, and middle satellite bands. Based on the number and location of the bands, the tested eight species can be divided into three groups: Group 1 comprisesE. sagittatum, with only 15 C-bands; Group 2, E. alpinum and E. pubigerum native to Germany, with 18 C-bands; Group 3, E. acuminatum, E. simplicifolium, E. leptorrhizum, and E. davidii, with the C-band number from 20 to 22.


### 3.3. Peroxide Isozyme

The results showed that the POD isozyme zymographs of various leaves of the same accession are similar. For accurate comparison among accessions, the zymographs of all accessions were prepared on a single bio-gel plate (Figure6). Sixteen bands of POD isozyme (POD 1–16) were detected in the eleven species. Each species has its own characteristic bands, different from others, showing that the POD isozyme zymographs vary greatly among genusEpimedium. Among the eleven species, the zymograph bands of E. pubigerum were the most abundant, with eleven bands (POD1–3, POD 7, and POD 10–16), E. yinjiangense with only one band (POD 1), and E. luodianense with two bands (POD 1 and 5). All the zymographs can be divided into three regions, namely, slow (Rf⩽0.20), medium (0.2<Rf⩽0.60), and fast (0.6<Rf⩽1.00).The POD isozyme zymogram and idiogram of twelve species of genusEpimedium. 1 = PU1, 2 = PU2, 3 = AL1, 4=AL2, 5 = AC37, 6 = AC22, 7 = LE10, 8 = LE5, 9 = LE6, 10 = WU15, 11 = WU10, 12 = WU16, 13 = FR1, 14 = SI1, 15 = SI2, 16 = YI1, 17 = YI2, 18 = LU1, 19 = LU4, 20 = MY1, 21 = SA1, and 22 = SA2.
(a)
(b)The dissimilarity coefficient (d) of isozyme zymograph is the symbol of relationships among species [27]. To find out the relationship of these species, the d and d¯ value of POD isozyme zymogram were studied and filled in Table 3. The d value of E. acuminatum and E. sagittatum is the lowest, 0.091, and E. franchetii and E. yinjiangense the highest, 1.00. The d¯ values of E. franchetii, E. pubigerum,and E. yinjiangense are higher, respectively, 0.678, 0.669, and 0.621. And the d¯ values of E. sagittatum and E. myrianthum (small-flower), respectively, are 0.432 and 0.418, lower than the others.Table 3
The interspecific dissimilarity coefficient (d, %) of POD zymogram of eleven barrenwort species and their means.
Species
FR
PU
YI
LE
LU
SI
AC
AL
WU
MY
SA
Mean (d-)
FR
67.8
PU
76.5
66.9
YI
100
83.3
62.1
LE
81.8
37.5
66.7
58.4
LU
75.0
86.4
33.3
71.4
57.0
SI
77.8
71.4
50.0
50.0
60.0
47.1
AC
50.0
64.7
71.4
63.6
50.0
33.3
45.3
AL
75.0
69.2
33.3
42.9
50.0
20.0
50.0
44.6
WU
45.5
62.5
66.7
60.0
42.9
50.0
27.2
42.9
44.3
MY
77.8
57.1
50.0
50.0
60.0
33.3
33.3
20.0
25.0
43.2
SA
63.6
62.5
66.7
60.0
42.9
25.0
9.1
42.9
20.0
25.0
41.8
Mean (d-)
67.8
66.9
62.1
58.4
57.0
47.1
45.3
44.6
44.3
43.2
41.8
52.59


### 3.4. HPLC Fingerprint of Flavonoids

The flavonoids HPLC fingerprints of twenty-one barrenwort accessions, belonging to eight species were observed (Figure7). The result showed that the HPLC fingerprint of different species were different. Each species had characteristic peaks, which were different from those seen in other species. All the HPLC fingerprint peaks can be divided into two regions: Region 1, retention time < 10 min, and Region 2, retention time > 10 min. Based on the number and location of peaks held by the species in Region 2, the eight species can be grouped into three types: Type 1 (E. sagittatum) with Peak 1–8, Type 2 (E. acuminatum, E. yinjiangense, E. wushanense, and E. simplicifolium) with Peak 1–5, and Type 3 (E. luodianense and E. leptorrhizum) without any peak, suggesting that the relation among E. acuminatum, E. yinjiangense, E. wushanense, E. simplicifolium, and E. davidii  is near each other; E. luodianense and E. leptorrhizum are also near. E. sagittatum is distant from other species.HPLC fingerprint of eight species. 1:E. wushanense. 2: E. sagittatum. 3: E. yinjiangense. 4: E. leptorrhizum. 5: E. simplicifolium. 6: E. luodianense. 7: E. davidii. 8: E. acuminatum.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


### 3.5. Interspecific Crossability

The crossabilities of fourteen accessions belonging to seven species were studied in the aspect of the grain set rate of hybridization (Table4). Results showed that the hybridizations of any combination among seven species are successful, and the rate of grain set among hybridization combinations vary obviously for the grain set rate of E. luodianense and E. letorrhizum is the highest (up to 92.44%) andE. myrianthum and E. acuminatum the lowest (only 15.38%). Based on the rates, seven species studied can be divided into two groups: Group one is only one species, E. myrianthum, with lower grain set rate of crossing with other species (almost all under 30%); Group two is the rest, with higher grain set rate (almost all over 30%).Table 4
The rate of grain set (%) of hybridization between species.
♂
♀
E. myrianthum
E. acuminatum
E. wushanense
E. letorrhizum
E. luodianense
E. simplicifolium
E. yinjiangense
E. myrianthum
15.38
15.83
26.97
35.82
16.85
30.09
E. acuminatum
29.17
49.51
24.34
47.06
73.35
41.67
E. wushanense
33.03
60.32
28.26
63.41
92.23
64.04
E. letorrhizum
28.69
29.62
42.86
92.44
87.01
76.47
E. luodianense
35.96
43.88
80.15
67.68
73.08
88.07
E. simplicifolium
35.05
71.43
88.78
75.00
48.20
77.01
E. yinjiangense
20.80
43.08
72.73
41.38
83.33
62.92Results also showed that there are no various differences between the grain set rates of positive and negative hybridization, suggesting that the significance of grain set rats of positive and negative hybridization in symboling the interspecific relationship of this genus is the same. So the means of grain set rats of positive and negative hybridization were analyzed by the unweighted pair-group method (UPGMA) (Figure8) to find the relationship of seven species researched in this paper. Results showed that E. myrianthum is very distant from other six species, and the relation between E. luodianense and E. yinjiangense is the nearest, and they are nearer related to E. letorrhizum.Figure 8
Clustering of seven species by the grain set rate of hybridization.


## 3.1. Morphological Observation and Karyotype Analyses of Chromosomes

Based on the size of flower, twelve species can be grouped into two types, type one is small flower includingE. sagittatumand E. myrianthum,type two is large flower, the rest. The results of chromosomal observations of twelve species showed all species studied were diploid (2n=2x=12) and had one pair of middle satellite chromosomes. Expect for the middle satellite chromosomes of E. leptorrhizum, E. simplicifoliumand E. wushanense are in short arms of no. 2 chromosomes; the rest are no. 1 chromosomes. Tetraploid cells sporadically were found in E. letorrhizum root meristems among twelve species. The total length of haploid complement (TCL) of E. yinjiangenseand E. alpinum are obviously small, 29.23±2.36 μm and 32.96±1.34 μm, respectively. And the range of chromosome length of E. yinjiangense is 2.15~2.92 μm and E. alpinum 2.22~3.06 μm.The parameters (Table2), figures (Figure 1), and karyotypes (Figure 2) of twelve species metaphase chromosomes were studied. The results showed that their karyotypes are similar to each other, only including m and sm two types chromosome. Twelve species karyotypes are all 2A type of Stebbin’s asymmetry categories. Among them, the karyotype formulas of E. sagittatum, E. myrianthum, E. acuminatun, E. wushanense, E. simplicifolium, E. yinjianense, E. luodianense, E. davidii, E. pubigerum, E. franchetii, andE. alpinum are 2n=2x=6m (2SAT) + 6sm, and E. leptorrhizumis 2n=2x=8m (2SAT) + 4sm.Table 2
Karyotype formula (KF), satellite position (SAT), total length of the haploid complement (TCL), range of chromosome length (range), intrachromosome asymmetry index (A1), interchromosome asymmetry (A2), mean centromeric index (CI), and Stebbin’s asymmetry categories (ST) of the studied Epimedium species.
Species
KF
SAT
TCL ± SE
Range
A
1
A
2
CI ± SE
ST
E. acuminatum
4m + 6sm + 2m (SAT)
1s
45.23
±
0.78
3.08~4.77
0.38
0.17
37.54
±
0.15
2A
E. yinjiangense
4m + 6sm + 2m (SAT)*
1s
29.23
±
2.36
2.15~2.92
0.43
0.14
35.76
±
0.28
2A
E. luodianense
4m + 6sm + 2m (SAT)*
1s
47.88
±
1.24
2.28~4.87
0.38
0.17
36.69
±
0.31
2A
E. leptorrhizum
6m + 4sm + 2m (SAT)*
2s
46.00
±
1.04
3.06~4.44
0.34
0.14
39.00
±
0.14
2A
E. simplicifoilum
4m + 6sm + 2m (SAT)*
2s
45.43
±
2.01
3.05~4.17
0.42
0.11
36.06
±
0.52
2A
E. wushanense
4m + 6sm + 2m (SAT)*
2s
46.86
±
0.88
3.33~4.44
0.33
0.10
39.40
±
0.46
2A
E. alpinum
4m + 6sm + 2m (SAT)
1s
32.96
±
1.34
2.22~3.06
0.35
0.13
38.39
±
0.39
2A
E. davidii
4m + 6sm + 2m (SAT)*
1s
47.32
±
1.67
3.33~4.72
0.42
0.16
36.08
±
0.22
2A
E. pubigerum
4m + 6sm + 2m (SAT)
1s
38.59
±
0.96
2.50~3.61
0.32
0.14
39.88
±
0.18
2A
E. franchetii
4m + 6sm + 2m (SAT)*
1s
38.04
±
0.82
2.91~4.72
0.36
0.18
38.20
±
0.29
2A
E. myrianthum
4m + 6sm + 2m (SAT)*
1s
45.45
±
1.27
3.38~4.15
0.40
0.08
36.69
±
0.38
2A
E. sagittatum
4m + 6sm + 2m (SAT)
1s
41.88
±
1.01
3.27~4.68
0.34
0.18
38.69
±
0.13
2A
SE: standard error, m: metacentric, sm: submetacentric, st: subteleocentric, s: short arm, and *indicates first description of species.Figure 1
Somatic chromosome ofEpimedium, all species with 2n = 12. 1–10: large-flower group. 1: E. acuminatum. 2: E. yinjiangense. 3: E. luodianense. 4: E. leptorrhizum. 5: E. simplicifolium. 6: E. wushanense. 7: E. alpinum. 8: E. davidii. 9: E. pubigerum. 10: E. franchetii. 11-12: small-flower group. 11: E. myrianthum. 12: E. sagittatum. Scale bar = 2 μm.Figure 2
Karyotypes of twelve species metaphase chromosomes, all species with 2n = 12. 1–10: large-flower group. 1:E. acuminatum. 2: E. yinjiangense. 3: E. luodianense. 4: E. leptorrhizum. 5: E. simplicifolium. 6: E. wushanense. 7: E. alpinum. 8: E. davidii. 9: E. pubigerum. 10: E. franchetii. 11-12: small-flower group. 11: E. myrianthum. 12: E. sagittatum. Scale bar = 2 μm.


## 3.2. Giemsa C-Band Analyses of Chromosomes

Eight species,E. sagittatum, E. alpinum, E. pubigerum, E. acuminatum, E. simplicifolium, E. wushanense, E. letorrhizum, and E. davidii, were treated by chromosomes C-banding. The metaphase chromosomes of C-banding, Giemsa C-banding karyotypes and idiograms of C-banding karyotype can be seen in Figures 3, 4, and 5, respectively.Figure 3
The metaphase chromosomes C, banding in the eight species of genusEpimedium. 1: E. simplicifolium. 2: E. leptorrhizum. 3: E. wushanense. 4: E. alpinum. 5: E. davidii. 6: E. acuminatum. 7: E. pubigerum. 8: E. sagittatum. Scale bar = 2 μm.Figure 4
Giemsa C-banded karyotypes in the eight species of genusEpimedium. 1: E. simplicifolium. 2: E. leptorrhizum. 3: E. wushanense. 4: E. alpinum. 5: E. davidii. 6: E. acuminatum. 7: E. pubigerum. 8: E. sagittatum. Scale bar = 2 μm.Figure 5
Idiograms of C-banded karyotypes in the eight species of genusEpimedium. 1: E. simplicifolium. 2: E. leptorrhizum. 3: E. wushanense. 4: E. alpinum. 5: E. davidii. 6: E. acuminatum. 7: E. pubigerum. 8: E. sagittatum.Results showed that the chromosomes C-banding of eight species vary remarkably, with 15 to 22 bands, consisting of centromeric bands, intercalary bands, terminal bands, and middle satellite bands. Based on the number and location of the bands, the tested eight species can be divided into three groups: Group 1 comprisesE. sagittatum, with only 15 C-bands; Group 2, E. alpinum and E. pubigerum native to Germany, with 18 C-bands; Group 3, E. acuminatum, E. simplicifolium, E. leptorrhizum, and E. davidii, with the C-band number from 20 to 22.


## 3.3. Peroxide Isozyme

The results showed that the POD isozyme zymographs of various leaves of the same accession are similar. For accurate comparison among accessions, the zymographs of all accessions were prepared on a single bio-gel plate (Figure6). Sixteen bands of POD isozyme (POD 1–16) were detected in the eleven species. Each species has its own characteristic bands, different from others, showing that the POD isozyme zymographs vary greatly among genusEpimedium. Among the eleven species, the zymograph bands of E. pubigerum were the most abundant, with eleven bands (POD1–3, POD 7, and POD 10–16), E. yinjiangense with only one band (POD 1), and E. luodianense with two bands (POD 1 and 5). All the zymographs can be divided into three regions, namely, slow (Rf⩽0.20), medium (0.2<Rf⩽0.60), and fast (0.6<Rf⩽1.00).The POD isozyme zymogram and idiogram of twelve species of genusEpimedium. 1 = PU1, 2 = PU2, 3 = AL1, 4=AL2, 5 = AC37, 6 = AC22, 7 = LE10, 8 = LE5, 9 = LE6, 10 = WU15, 11 = WU10, 12 = WU16, 13 = FR1, 14 = SI1, 15 = SI2, 16 = YI1, 17 = YI2, 18 = LU1, 19 = LU4, 20 = MY1, 21 = SA1, and 22 = SA2.
(a)
(b)The dissimilarity coefficient (d) of isozyme zymograph is the symbol of relationships among species [27]. To find out the relationship of these species, the d and d¯ value of POD isozyme zymogram were studied and filled in Table 3. The d value of E. acuminatum and E. sagittatum is the lowest, 0.091, and E. franchetii and E. yinjiangense the highest, 1.00. The d¯ values of E. franchetii, E. pubigerum,and E. yinjiangense are higher, respectively, 0.678, 0.669, and 0.621. And the d¯ values of E. sagittatum and E. myrianthum (small-flower), respectively, are 0.432 and 0.418, lower than the others.Table 3
The interspecific dissimilarity coefficient (d, %) of POD zymogram of eleven barrenwort species and their means.
Species
FR
PU
YI
LE
LU
SI
AC
AL
WU
MY
SA
Mean (d-)
FR
67.8
PU
76.5
66.9
YI
100
83.3
62.1
LE
81.8
37.5
66.7
58.4
LU
75.0
86.4
33.3
71.4
57.0
SI
77.8
71.4
50.0
50.0
60.0
47.1
AC
50.0
64.7
71.4
63.6
50.0
33.3
45.3
AL
75.0
69.2
33.3
42.9
50.0
20.0
50.0
44.6
WU
45.5
62.5
66.7
60.0
42.9
50.0
27.2
42.9
44.3
MY
77.8
57.1
50.0
50.0
60.0
33.3
33.3
20.0
25.0
43.2
SA
63.6
62.5
66.7
60.0
42.9
25.0
9.1
42.9
20.0
25.0
41.8
Mean (d-)
67.8
66.9
62.1
58.4
57.0
47.1
45.3
44.6
44.3
43.2
41.8
52.59


## 3.4. HPLC Fingerprint of Flavonoids

The flavonoids HPLC fingerprints of twenty-one barrenwort accessions, belonging to eight species were observed (Figure7). The result showed that the HPLC fingerprint of different species were different. Each species had characteristic peaks, which were different from those seen in other species. All the HPLC fingerprint peaks can be divided into two regions: Region 1, retention time < 10 min, and Region 2, retention time > 10 min. Based on the number and location of peaks held by the species in Region 2, the eight species can be grouped into three types: Type 1 (E. sagittatum) with Peak 1–8, Type 2 (E. acuminatum, E. yinjiangense, E. wushanense, and E. simplicifolium) with Peak 1–5, and Type 3 (E. luodianense and E. leptorrhizum) without any peak, suggesting that the relation among E. acuminatum, E. yinjiangense, E. wushanense, E. simplicifolium, and E. davidii  is near each other; E. luodianense and E. leptorrhizum are also near. E. sagittatum is distant from other species.HPLC fingerprint of eight species. 1:E. wushanense. 2: E. sagittatum. 3: E. yinjiangense. 4: E. leptorrhizum. 5: E. simplicifolium. 6: E. luodianense. 7: E. davidii. 8: E. acuminatum.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


## 3.5. Interspecific Crossability

The crossabilities of fourteen accessions belonging to seven species were studied in the aspect of the grain set rate of hybridization (Table4). Results showed that the hybridizations of any combination among seven species are successful, and the rate of grain set among hybridization combinations vary obviously for the grain set rate of E. luodianense and E. letorrhizum is the highest (up to 92.44%) andE. myrianthum and E. acuminatum the lowest (only 15.38%). Based on the rates, seven species studied can be divided into two groups: Group one is only one species, E. myrianthum, with lower grain set rate of crossing with other species (almost all under 30%); Group two is the rest, with higher grain set rate (almost all over 30%).Table 4
The rate of grain set (%) of hybridization between species.
♂
♀
E. myrianthum
E. acuminatum
E. wushanense
E. letorrhizum
E. luodianense
E. simplicifolium
E. yinjiangense
E. myrianthum
15.38
15.83
26.97
35.82
16.85
30.09
E. acuminatum
29.17
49.51
24.34
47.06
73.35
41.67
E. wushanense
33.03
60.32
28.26
63.41
92.23
64.04
E. letorrhizum
28.69
29.62
42.86
92.44
87.01
76.47
E. luodianense
35.96
43.88
80.15
67.68
73.08
88.07
E. simplicifolium
35.05
71.43
88.78
75.00
48.20
77.01
E. yinjiangense
20.80
43.08
72.73
41.38
83.33
62.92Results also showed that there are no various differences between the grain set rates of positive and negative hybridization, suggesting that the significance of grain set rats of positive and negative hybridization in symboling the interspecific relationship of this genus is the same. So the means of grain set rats of positive and negative hybridization were analyzed by the unweighted pair-group method (UPGMA) (Figure8) to find the relationship of seven species researched in this paper. Results showed that E. myrianthum is very distant from other six species, and the relation between E. luodianense and E. yinjiangense is the nearest, and they are nearer related to E. letorrhizum.Figure 8
Clustering of seven species by the grain set rate of hybridization.


## 4. Discussion

### 4.1. Generic Evolution

The results showed that karyotypes of twelve species are 2A symmetry type of Stebbins [22] standard and similar to each other only with m and sm chromosomes, and interspecific hybridization easily succeeds, suggesting that Epimedium is a very conservative genus in the process of evolution.The longer the evolutionary history, the larger the DNA differentiation and the higher the average value of zymogram distance [27]. Su and Liu [28] concluded that Dysosmais an age-old genus based on the average value of its isozyme zymogram distance, 0.828. In our study, this value was 0.526, indicating that Epimediummay be a more ancient genus in the family Berberidaceae. This result is consistent with those studies results of geobiology [6], pollen morphology [8], molecular biology [13–16], and cytology [23, 29] of this genus, also supported by the deduction about the phylogeny of Berberidaceae by Wu et al.  [2].Karyotype formulas ofE. sagittatum, E. myrianthum, E. acuminatum, E. wushanense, E. simplicifolium, E, yinjiangense, E. luodianense, E. davidii, E. pubigerum, E. franchetii, and E. alpinum are 2n = 2x = 6m (2SAT) + 6sm, and E. leptorrhizum is 2n = 2x = 8m (2SAT) + 4sm. Karyotype formulas of four genus, Diphylleia, Dysasma, Podophyllum and Sinopodophyllum, of family Berberidaceae are 2n = 2x = 8m (SAT) + 2st + 2t, 2n = 2x = 8m (2SAT) + 2st (SAT) + 2t, 2n = 2x = 6m (4SAT) + 2sm + 2st + 2t, and 2n = 2x = 8m (2SAT) + 2st (2SAT) + 2t, respectively   [23, 29]. Along with the level of evolution advancement, the asymmetry of karyotype will enhance in family Berberidaceae [29]. Obviously, the karyotypes of Epimedium are the most symmetrical among genus of Berberidaceae, suggesting that Epimedium is the most original genus in the family Berberidaceae.


### 4.2. Interspecific Relationship

Results of chromosomes C-banding, POD isozyme, HPLC fingerprint, and interspecific hybridization researches all show that small-flower species,E. sagittatum and E. myrianthum, are distant from the ten large flower species, strongly supporting the flower size which is one of the important taxonomy symbols of this genus, and genus Epimedium should be classed into two sections: small and large flower.In large flower species, the relation amongE. leptorrhizum, E. luodianense,andE. jinjiangenseis near based on results of POD isozyme, HPLC fingerprint, and the grain set rate of interspecific hybridization. Results of HPLC fingerprint that showed peaks in Region 2 of E. acuminatum, E. simplicifolium, E. wushanense, and E. davidii are the same with Peaks 1–5, suggesting that the relation of these four species are near, also supported by the result of interspecific hybridization research. Results of chromosomes C-banding showed that E. alpinumandE. pubigerum were divided into the same group, suggesting that the relation of these two species are near. Results of POD isozyme analyses showed that the zymograph of E. franchetii varies greatly from the others, suggesting that E. franchetiiis distant from the rest.Modernistic taxonomy sparkplug that species set by classical morphologic criteria should be classed into biological species renewedly. Results of interspecific hybridization among seven barrenwort species showed onlyE. myrianthum, with lower grain set rate of crossing with other species (almost all under 30%), maybe a biological species. However, the others are not biological species for there are high grain set rate each other. There will be lots of works to do in studying biological species of this genus.


### 4.3. Origin of This Genus

The distribution pattern ofEpimedium is the typical type of old world temperate zone [6], internationally distributing in three discontinuous areas of the Mediterranean Sea and Western Asia, China, and Japan. China is the diversity centre of this genus with about 50 species and five infraspecies on record [25], mostly occurring in the south-western China. However, studies on the origin of this genus still have not sure conclusions.This study on POD isozyme showed that the means ofd  values of the two species of the small-flowers group, E. sagittatumand E. myrianthum, were 0.432 and 0.418, respectively, lower than the large-flowers group species, implying that the small-flowers group is older in genus Epimedium. It is possible that the course of evolution of Epimediumwas from the small-flowers group to the large-flowers group. Since it is known that south-western China is the origin centre of the small-flowers group of this genus [6], south-western China may also be the origin centre of the genus Epimedium.


## 4.1. Generic Evolution

The results showed that karyotypes of twelve species are 2A symmetry type of Stebbins [22] standard and similar to each other only with m and sm chromosomes, and interspecific hybridization easily succeeds, suggesting that Epimedium is a very conservative genus in the process of evolution.The longer the evolutionary history, the larger the DNA differentiation and the higher the average value of zymogram distance [27]. Su and Liu [28] concluded that Dysosmais an age-old genus based on the average value of its isozyme zymogram distance, 0.828. In our study, this value was 0.526, indicating that Epimediummay be a more ancient genus in the family Berberidaceae. This result is consistent with those studies results of geobiology [6], pollen morphology [8], molecular biology [13–16], and cytology [23, 29] of this genus, also supported by the deduction about the phylogeny of Berberidaceae by Wu et al.  [2].Karyotype formulas ofE. sagittatum, E. myrianthum, E. acuminatum, E. wushanense, E. simplicifolium, E, yinjiangense, E. luodianense, E. davidii, E. pubigerum, E. franchetii, and E. alpinum are 2n = 2x = 6m (2SAT) + 6sm, and E. leptorrhizum is 2n = 2x = 8m (2SAT) + 4sm. Karyotype formulas of four genus, Diphylleia, Dysasma, Podophyllum and Sinopodophyllum, of family Berberidaceae are 2n = 2x = 8m (SAT) + 2st + 2t, 2n = 2x = 8m (2SAT) + 2st (SAT) + 2t, 2n = 2x = 6m (4SAT) + 2sm + 2st + 2t, and 2n = 2x = 8m (2SAT) + 2st (2SAT) + 2t, respectively   [23, 29]. Along with the level of evolution advancement, the asymmetry of karyotype will enhance in family Berberidaceae [29]. Obviously, the karyotypes of Epimedium are the most symmetrical among genus of Berberidaceae, suggesting that Epimedium is the most original genus in the family Berberidaceae.


## 4.2. Interspecific Relationship

Results of chromosomes C-banding, POD isozyme, HPLC fingerprint, and interspecific hybridization researches all show that small-flower species,E. sagittatum and E. myrianthum, are distant from the ten large flower species, strongly supporting the flower size which is one of the important taxonomy symbols of this genus, and genus Epimedium should be classed into two sections: small and large flower.In large flower species, the relation amongE. leptorrhizum, E. luodianense,andE. jinjiangenseis near based on results of POD isozyme, HPLC fingerprint, and the grain set rate of interspecific hybridization. Results of HPLC fingerprint that showed peaks in Region 2 of E. acuminatum, E. simplicifolium, E. wushanense, and E. davidii are the same with Peaks 1–5, suggesting that the relation of these four species are near, also supported by the result of interspecific hybridization research. Results of chromosomes C-banding showed that E. alpinumandE. pubigerum were divided into the same group, suggesting that the relation of these two species are near. Results of POD isozyme analyses showed that the zymograph of E. franchetii varies greatly from the others, suggesting that E. franchetiiis distant from the rest.Modernistic taxonomy sparkplug that species set by classical morphologic criteria should be classed into biological species renewedly. Results of interspecific hybridization among seven barrenwort species showed onlyE. myrianthum, with lower grain set rate of crossing with other species (almost all under 30%), maybe a biological species. However, the others are not biological species for there are high grain set rate each other. There will be lots of works to do in studying biological species of this genus.


## 4.3. Origin of This Genus

The distribution pattern ofEpimedium is the typical type of old world temperate zone [6], internationally distributing in three discontinuous areas of the Mediterranean Sea and Western Asia, China, and Japan. China is the diversity centre of this genus with about 50 species and five infraspecies on record [25], mostly occurring in the south-western China. However, studies on the origin of this genus still have not sure conclusions.This study on POD isozyme showed that the means ofd  values of the two species of the small-flowers group, E. sagittatumand E. myrianthum, were 0.432 and 0.418, respectively, lower than the large-flowers group species, implying that the small-flowers group is older in genus Epimedium. It is possible that the course of evolution of Epimediumwas from the small-flowers group to the large-flowers group. Since it is known that south-western China is the origin centre of the small-flowers group of this genus [6], south-western China may also be the origin centre of the genus Epimedium.



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*Source: 271578-2013-11-19.xml*