Relationships among six herbal species ( Curcuma ) assessed by four isozymes

Resumen. Se estudiaron cuatro isoenzimas [superóxido dismutasa (SOD), polifenol oxidasa (PPO), málico deshidrogenasa (MDH) y citocromo oxidasa (COD)] para identificar seis especies herbáceas de Curcuma L. Los 37 especimenes estudiados produjeron un total de 168 bandas de isoenzimas polimórficas. Los coeficientes de distancia genética (GS) variaron entre 0.08 y 0.54. El dendrograma, obtenido de acuerdo a las bandas de isoenzimas polimórficas por el método UPGMA con el software NTSYS-pc2.1, contribuyó a resolver la filogenia. A partir del dendrograma, fue posible diferenciar entre los especímenes silvestres y cultivados de C. longa, y dentro de las especies de C. sichuanensis.


INTRODUCTION
Curcuma L. (Zingiberaceae) is widespread in the tropics of Asia, Africa and Australia, and it is composed of approximately 70 species (Purseglove, 1974).About 10 Curcuma species are distributed in China (Xiao et al., 1997;Li et al., 2001;Ye et al., 2008).Six of those species have been used as Chinese herbal medicine for more than a thousand years.For example, an extract of their rhizomes exhibits anti-inflammatory, anticancer activity (Moussavi et al., 2006).There are three traditional Chinese medicines [Radix Curcumae (also named Yujin), Rhizoma Curcumae Longae (also named Jianghuang) and Rhizoma Curcumae (also named Ezhu)] derived from these six Curcuma species.Roots (Chen, 1981;Zhu, 1992).
In Traditional Chinese Medicine (TCM) the same medicinal substances can be produced from these six Curcuma species, although one of them can be used as a different medicinal substance.Morphological characteristics are very large for rhizomes and leaves, both intra-and inter-species.The similarities of the growth habit, leaf-shapes, and flowers among the study Curcuma species are so great that it is generally difficult to distinguish the species at both the vegetative and reproductive stages.The Curcuma flowering season vary from April to October, and it is common that the same species has flowers with different colors.These problems have been troublesome in phylogenetic analysis, and made the clinic of TCM inaccurate.However, the correct identity is important to confirm the sources of origin of herbal drugs within the genus Curcuma (Sasaki et al., 2002;Cao & Katsuko, 2003).
Different techniques have different advantages and disadvantages.It is then necessary to confirm the origin of and the genetic relationships among these six herbal species by various methods.Because of the effectiveness of molecular markers in plant systematics (Crawford, 1991), the isozymic technique has been widely applied to (1) deal with evolvement of botany, (2) identify idioplasm resources (Apavatrut et al., 1999;Monireh, 2007), and (3) investigate genetic relationships (Fang et al., 1993;Guo & Li, 2000;Wu et al., 2002;Arzate-Fernandez et al., 2005).
The objectives of this paper were to (1) evaluate the phylogenetic relationships among Curcuma herbal species; (2) explore the taxonomic status of C. sichuanensis and C. chuanhuangjiang species; and (3) identify the origin of traditional medicine in China.

MATERIALS AND METHODS
Plant materials.Thirty seven specimens of the genus Curcuma, which were divided into six species, were analyzed in this study (Table 1).Thirty one specimens were collected from different localities in the Sichuan province, and the other 6 specimens were gathered from the Guangxi Medicinal Botanical Garden.Sichuan and Guangxi belong to well-known regions of these species, and Sichuan is the geo-herbalism habitat of C. longa, C. sichuanensis, C. phaeocaulis and C. chuanhuangjiang in China (Hu, 1998).
Protein extraction and isozyme analysis.For protein extraction, 0.5 g of tender leaves were powdered in liquid nitrogen.Flour of the leaves was first homogenized in the PBS (0.5 mol, pH 7.8) using 1:1 (v/v) ratio, and then centrifuged at -4 °C for 12 minutes at 12000 rpm.The supernatant was collected and stored at -20 °C.PAGE was performed according to a modified method of using vertical slab gels (1.5 mm thick) and was set up forming a discontinuous system of two layers.These two layers were: (i) resolving gel: 13.5 cm layer of 7.5% polyacrylamide, and (ii) stacking gel: 1.5 cm layer of 3% polyacrylamide.Four isozymes were tested and the staining protocols followed Wendel & Weeden (1989).Only clear isozyme bands were scored (numbered beginning with the running closer to the origin) and enzymatic schema diagrams painted according to RF values (relative mobility) (Kuhns & Fretaz, 1978).The frequency distribution of isozyme bands was calculated according to enzymatic schema diagram (Table 2 -5).Different patterns occurring in each zone of activity (not single bands data) were scored as discrete variables, using '1' to indicate presence, and '0' to indicate absence of a unique pattern.A dendrogram, that depicts the degree of relationships among the taxa, was produced using hierarchical cluster analysis [NTSYS-pc2.1 software (Rohlf, 2000), UPGMA method (Sneath & Sokal, 1973)].

RESULTS
Isozyme bands.All 37 specimens produced a total of 168 polymorphism isozyme bands; of these, there were 58 PPO, 47 COD, 28 SOD and 35 MDH.Portion pictures of electrophoresis and schema graphs are shown in Figs 1 to 5. Variation of isozyme bands in each specimen were 6-13 (PPO), 6-12 (COD), 2-8 (SOD), and 0-8 (MDH).The isozyme bands showed polymorphism.Consequently, the four isozymes patterns were suitable for fingerprints to distinguish different species of the genus Curcuma.
The bands presented large diversities between species.Genetic relationships analysis.The Jaccard's similarity coefficients were calculated with the four isozymes data and the genetic distance coefficients (GS) varied from 0.08 to 0.54 among the 37 specimens.The phylogenetic tree (Fig. 6) was constructed following the UPGMA method according to RF values.
When GS was 0.52, the 37 specimens were largely divided into two groups.The first group contained 18 specimens of C. longa, all of which were collected from the Sichuan province.The second group included four specimens of C. longa, and all specimens of the other five species.In the first group, most of the cultivated C. longa specimens assembled together as a subgroup and some of the wild C. longa clustered as another subgroup.

DISCUSSION
When GS was at 0.28, C. kwangsiensis clustered together with one specimen of C. phaeocaulis (number 33); thereafter, they grouped with the other four specimens of C. phaeocaulis.Results indicated that C. kwangsiensis and C. phaeocaulis had relatively closer genetic relationships.However, there might be an error due to the phylogeny tree methods, or this error may be the result of the long-term companion planting in the Medicinal Botanical Garden of Guangxi Autonomous Region.Further DNA analyses are needed to detect this error.
On the study of RAPD marker analysis (Chen et al., 1999), 119 bands were produced with 12 primers, and the genetic distance was 0. 164 between C. wenyujin and C. sichuanensis; the intraspecific genetic distance was much larger than interspecific: they were 0.866 for C. wenyujin and 0.885 for C. sichuanensis.Chen et al. recognized that C. wenyujin was close to C. sichuanensis, and merged C. sichuanensis into C. wenyujin.It is difficult to identify these two species on the level of DNA.While the 18S rRNA and trnK gene sequences of C. sichuanensis and C. longa corresponded completely to the types either 1a or 1b (Sasaki et al., 2002), and the sequence of C. wenyujin belonged to type 5, great differences were shown between C. wenyujin and C. sichuanensis in the trnK and 18S rRNA sequences.C. sichuanensis and C. wenyujin clustered together after clustering study of leaf epidermal features (Xiao et al., 2000).However, Xiao et al. confirmed that C. sichuanensis was close to C. longa rather than C. wenyujin following investigation of the origin and the colour of tuber.It means that former studies of these two species had some different results.Based on our study, the isozymes patterns of PPO, COD, SOD, and MDH showed significant diversities within Deng JB et al., FYTON 80 (2011) Fig. 2. Isozyme patterns of PPO enzyme to identify the 37 specimens of six Curcuma species.(Numbers from 1 to 37 refer to the materials listed in Table 1).Fig. 2. Modelo de distribución de las isoenzimas de la enzima PPO para identificar los 37 especímenes de las seis especies de Curcuma.(Los números 1 a 37 se refieren a los materiales listados en la Tabla 1).
Fig. 3. Isozyme patterns of COD enzyme to identify the 37 specimens of six Curcuma species.(Numbers from 1 to 37 refer to the materials listed in Table 1).
Fig. 4. Isozyme patterns of SOD enzyme to identify the 37 specimens of six Curcuma species.(Numbers from 1 to 37 refer to the materials listed in Table 1).
Fig. 5. Isozyme patterns of MDH enzyme to identify the 37 specimens of six Curcuma species.(Numbers from 1 to 37 refer to the materials listed in Table 1).The relationship between C. longa and C. sichuanensis was complex (Xia et al., 1999;Xiao et al., 1997Xiao et al., , 2000Xiao et al., , 2001)).On the morphological study of leaves and rhizomes, Xiao et al. (2004aXiao et al. ( , 2004bXiao et al. ( , 2004c) )   Although a detailed Flora Sichuanica by Zhu (1992), C. chuanhuangjiang is not mentioned in the Flora of China.Liu & Wu (1999) merged C. chuanhuangjiang into C. kwangsiensis.Xiao et al. (2004aXiao et al. ( , 2004bXiao et al. ( , 2004c) thought that C. chuanhuangjiang was the cultivated mutation of C. longa.In our study, the isozymes patterns of C. chuanhuangjiang were dissimilar to the other 36 specimens.Taking into account the previous analysis of Cao & Katsuko (2003) and Tang et al (2008), we believed that it is much more reasonable to retain C. chuanhuangjiang as an individual species.
In conclusion, the four isozymes successfully supported the taxonomical classification of the six Curcuma species.From the dendrogram, 3/4 of the wild specimens of C. longa (numbers 11-18), and the two wild species of C. sichuanensis (numbers 25 and 26), clustered together first in groups I and II, and then gathered with other cultivated specimens; it is shown that the protein differentiation already occurred between cultivated and wild species.We strongly suggest paying attention to the distinction between cultivated and wild specimens when making classification, as well as on the clinic of TCM.
indicated that C. sichuanensis was the cultivated variety of C. longa.However, they contradicted themselves in their study of leaves and rhizomes: (i) on the morphological study of leaves, C. wenyujin and C. sichuanensis clustered together firstly, and C. longa was far away from them; (ii) on the morphological study of rhizomes, C. longa and C. sichuanensis got together at first.Quan et al. (2005) examined the contents of curdione and turmerol by means of HPLC and 5sRNA sequence on five species (C.kwangsiensis, C. wenyujin, C. phaeocaulis, of C. longa L.; C. wenyujin Y. H. Chen et C. Ling; C. kwangsiensis S. G. Lee et C. F. Liang, and C. phaeocaulis Valeton (used as herbal species) are officially recorded in Chinese Pharmacopoeia (2010).However, roots of C. sichuanensis C. K. Hsich et H. Zhang, and C. chuanhuanjiang Z. Y. Zhu can also be used as Radix Curcumae; also, the rhizomes of C. chuanhuanjiang, C. sichuanensis and C. wenyujin can be used as Rhizoma Curcumae Longae in folk therapeutic uses

Table 1 .
Origin of the study materials in this research.Origen de los materiales estudiados en esta investigación.

Table 2 .
In the second group, four cultivated C. longa specimens and five C. sichuanensis specimens (numbers 23-27) belonged to the same subgroup; another subgroup included all the specimens of C. phaeocaulis, C. chuanhuangjiang, C. kwangsiensis, C. wenyujin and two cultivated C. sichuanensis species (numbers 28 and 29).Rf value and frequency distribution of PPO bands.

Table 3 .
Rf value and frequency distribution of COD bands.

Table 4 .
Rf value and frequency distribution of SOD bands.

Table 4 .
Valores de Rf y frecuencia de distribución de bandas SOD.

Table 5 .
Rf value and frequency distribution of MDH bands.
Tang et al. (2008)n UPGMA analysis of genetic similarity obtained from isozymes data.(Numbersfrom 1 to 37 refer to the materials listed in Table1).Tang et al. (2008)recognized that C. sichuanensis was the cultivated mutation species of C. longa by isozyme patterns of POD and EST.In the present study, three cultivated (numbers 23, 24 and 27) and two wild specimens (numbers 25 and 26) of C. sichuanensis were clustered together with four specimens of C. longa at first; two other cultivated specimens (numbers 28 and 29) of C. sichuanensis were gathered with C. chuanhuangjiang, C. phaeocaulis, C. kwangsiensis and C. wenyujin.No doubt that the taxonomic status of C. sichuanensis needs further study.
C. longa, C. sichuanensis).Their results showed that C. longa was on intimate relationship with C. sichuanensis.