Analysis of the Sequence of ITS1-5.8S-ITS2 Regions of the Three Species of Fructus Evodiae in Guizhou Province of China and Identification of Main Ingredients of Their Medicinal ChemistryIdentification of Main Ingredients of Their Medicinal Chemistry

A total of fourteen specimens of three Fructus Evodiae species were collected from Zunyi and Tongren of Guizhou Province in China. The internal transcribed spacer (ITS) region (ITS1, ITS2 and 5.8S rDNA) of the nuclear ribosomal DNA (nrDNA) of the fourteen species were amplified and sequenced and another two sequences of Evodia rutaecarpa (JUSS.) BENTH species were obtained from Hunan Academy of Traditional Chinese Medicine, Changsha, Hunan Province of China. In addition to, the main ingredients of their medicinal chemistry of all specimens were identified by high performance liquid chromatography (HPLC). The three different morphological Fructus Evodiae species could not be distinctly classified by phylogenetic analysis of ITS combining the main ingredients of medicinal chemistry. This conclusion is inconsistent with early research results.


Introduction
Fructus Evodiae (synonyms: Tetradium ruticarpum), which is the dried nearly ripe fruit of Evodia rutaecarpa ( (Zhengyi et al., 2007). Fructus Evodiae cultivation has a long history in China Guizhou province, mainly distributed in Tongren, Zunyi , and other places. With its first-class quality, the output ranks first in China main exporting to Southeast Asia as Chinese traditional commodities.
In Pharmacopoeia of P. R. China (Part I), Fructus Evodiae is classified into three groups, EB; EBOH or EBBH (Chi, 2000), however, the latter two are considered ered as the variants of the former group. So far, this taxonomy is more acceptable to most scholars. As a medicine product on the market, according to the size of the flower of plants, Fructus Evodiae is also grouped into two sections, big flowers referred to EB and small flowers referred to EBOH or EBBH. In general, all above classifications are based on the individual characters portrayed and macrolevel observation. In practice, those classifications are facing challenges. For example, Fructus Evodiae is identified mainly relying on shapes of its leaves and fruit , but it has a strong seasonal limitations, for example, in winter and spring, its breeding and screening of seedlings face the identification difficulties. In addition, many unscrupulous traders often use the fake and shoddy Fructus Evodiae to cheat consumers due to identification difficulties. Those require that people must find a new classification and identification methods of Fructus Evodiae.
Eukaryotic ribosomal RNA genes (known as ribosomal DNA or rDNA) are found as parts of repeat units that are arranged in tandem arrays, ITS (for internal transcribed spacer) refers to a piece of non-functional RNA situated between structural ribosomal RNAs (rRNA) on a common precursor transcript. In the transcribed region, internal transcribed spacers (ITS) are found on either side of 5.8S rRNA gene and are described as ITS1 and ITS2. The length and sequences of ITS regions of rDNA repeats are believed to be fast evolving and therefore may vary. Universal PCR primers designed from highly conserved regions flanking the ITS and its relatively small size (600-700 bp) enable easy amplification of ITS region due to high copy number (up to-30000 per cell (Dubouzet and Shinoda 1999) of rDNA repeats). This makes the ITS region an interesting subject for evolutionary/phylogenetic investigations (Baldwin et al., 1995;Hershkovitz et al., 1996;Hershkovitz et al., 1999) as well as biogeographic investigations (Sharma et al., 1993;Suh et al., 1993;Hsiao et al., 1994;Dubouzet et al., 1999). A molecular approach, using sequence data from the internal transcribed spacer (ITS) of nuclear ribosomal DNA (nrDNA) were used in this study. The initial results of molecular phylogenetic analyses on taxa from the ITS region of Fructus Evodiae were showed in this paper. In addition, the content of Evodiamine and Rutaecarpine, both of which are major bioactive compounds in Fructus Evodiae, was determined by High Performance Liquid Chromatography (HPLC).  Evodiae, its specimens were grouped into 3 types, EB (shape-clustering and larger petals), EBOH (shape-dispersing and smaller petals, 13-18cm leaves with highly dense dots in diameter, commonly found in the limestone mountain), EBOH (shape-dispersing and smaller petals and about 10cm in diameter leaves with sparse dots in diameter, commonly found in relatively flat lands) (Zhengyi et al., 2007). All specimens were identified by Prof. Dr.Hesun.Zhi, Department of Clinical Biochemistry, Guiyang Medical College China 550004. Fresh leaves were collected, using ultra-low-temperature ice bags to conserve and keeping in the ultra-low-temperature refrigerator. Details of the plant materials are shown in Table 1.

DNA extraction, ITS amplification, cloning and sequencing
(a) DNA extraction and purification: Total cellular DNA was isolated using modified CTAB method based on the traditional one (Doyle et al., 1987). The isolated DNA was purified by RNase A treatment and chloroform: isoamyl alcohol extraction. The quality and quantity of DNA samples was checked on agarose gel using lambda DNA as marker. (c) Cloning and sequencing: Purified DNA was ligated in pMD18®-T Easy vector (TaKaRa Corp., China) overnight at 16 o C. The ligated DNA was transformed in DH5α competent cells. The recombinant clones were identified through blue/white color selection and the presence of insert in the recombinant clones (white colonies) was confirmed following colony PCR. For sequencing, plasmid DNA was isolated following alkali lysis method (Sambrook et al., 1989). The insert DNA was sequenced on contract using automated sequencing facility at TaKaRa Corp., China.
(e) Sequence submission: Sequences of clones were submitted directly to GenBank through Bankit (a World Wide Web sequence submission server available at NCBI home page). The sequences are available on line (http:// www.ncbi.nlm.nih.gov) and can be located by accession numbers or GI numbers EU663533-663546.
An initial alignment of the ITS sequences was generated using CLUSTALW (Thompson et al., 1994;Higgins et al., 1996 ), and then made several minor adjustments manually. The sequences with those published on the ITS of Fructus Evodiae (Dan et al., 2008;Poon et al., 2007) were compared to determine boundaries of ITS1ans ITS2. A total of 616 nucleotides were included for the phylogenetic analyses under maximum-parsimony (MP) criteria. Gaps were coded as missing data. MP phylogenetic analyses were accomplished using PAUP* 4.0b10 (Swofford, 2000) on PC with windows platform. Pairwise evolutionary distances between accessions were generated under the Kimura 2-parameter model (Kimura, 1980). Each nucleotide position was treated as an independent, unordered, multistate character of equal weight. All characters have equal weight and the parameter setting with weights transversions 10 times transitions. A parsimony heuristic search was performed using addition sequence set at random, with 1000 repetitions, ACCTRAN character state optimization, tree bisectionreconnection (TBR) branch swapping, and MULTRREES on, trees are unrooted. The ÷2-test of paup*4.0b4a (David, 1999) was performed to obtain the information about the homogeneity of the nucleotide distribution. The robustness and stability of parsimony trees were estimated by using bootstrapping with 100 replicates.

Result
The base composition in the dataset was balanced with a mean G+C content of 62.9% for the aligned complete sequences. Table 1 shows the distribution of nucleotides as well as the G+C content in ITS1+5.8S+ITS2 complete sequences. After exclusion of highly variable sites to minimize the risk of a phylogenetic tree construction based on nucleotide distribution (Saitou et al., 1989;Hasegawa et al., 1993;Steel et al., 1993) the distribution pattern resulted in a chi-square-test with p=1.0000, allowing for phylogenetic reconstruction.

Internal Transcribed Spacer Sequence Diversity
16 complete ITS sequences were generated for the 4 EB and 6 EBBOH and 6 EBBH species. Each full sequence included complete sequences of ITS1, the 5.8S rRNA gene, and ITS2. All complete 5.8S sequences were 163 nucleotides in length, except for the two sequences from EB.JS and EB.JL, which were 182 nucleotides long. The ITS1 sequences varied in length from 220 to 232bp, whereas the ITS2 sequences varied from 196 to 220 bp. Alignment of the ITS1 sequences required the hypothesis of 11 insertion/ deletion events of 1-2 bp each and resulted in an aligned length of 246 bp. Alignment of the ITS sequences required 3 indels of 1 bp each and 1 gap of 6 bp for a total aligned length of 617 bp. The guanine +cytosine (G+C) content averaged , respectively , 64.4%, 64.80% and 64.70% in ITS1, 54.62%, 54.09% and 53.89% in 5.8S, and 67.63%, 67.78% and 67.66% in the ITS in EB, EBBH and EBOH (shown in Table 2).
The studies show that some plant genomes harbor multiple, and in some cases, highly divergent ITS sequences, e.g., Buckler and Holtsford (1996), more than one clone for most studied accessions were sampled. When considering only substitution polymorphisms, the ITs sequence of EBBH.ZY1 was identical to EBOH.ZY2, EBBH.TR3 and EBBH were also identical as well because their evolutionary distance equals to zero (Table 3). Fourteen unique sequences among the 16 sequences included in the main phylogenetic analysis were selected in this study, 31 of 616 characters (5.03%) exhibited substitution polymorphisms, and 16 (2.3%) of those were parsimony-informative. These Informative sites were approximately equally distributed be-    Table 2). Over all, in the entire ITS region, 15 (2.43%) autapomorphy sites (variation at a same site in one sequence) were detected, similarly, 24 (3.9%) synapomorphic sites (variation at the same site in more than one sequence) were detected ( supplementary Fig 1 online).

Intraspecific Sequence Divergence in ITS1, ITS2 and 5.8S Regions
Of the three regions of the entire ITS, the sequence of 5.8S rRNA coding region, as expected, was conserved amongst EB.EBOH and EBBH species and this region exhibited only four substitutions (including three transitions and one transversion) and( supplementary Fig 1 online ). It suggested that a similar level of intraspecific sequence divergence in 5.8S region in the three species.
Four EB (4 sequences), six EBBH (6 sequences) and six EBOH (6 sequences) accessions were used to investigate intraspecific ITS variation. In EBBH, 5 of 6 sequences were unique, sequence divergence (Kimura 2-parameter distances) ranged from 0.000 to 0.023 (mean =0.012) ( Table  3); In EBOH, all six sequences were unique, sequence divergence ranged from 0.003 to 0.018 (mean =0.01); In EB, four sequences were unique, sequence divergence ranged from 0.005 to 0.027 (mean =0.0178). Therefore the three groups of species showed low within-group sequence divergence, it also clearly indicated that on the basis of se-quence divergence, due to substitutions in the ITS region, the EB genotypes were more diverse than the EBBH and EBOH genotypes. The mean sequence divergence (Kimura 2-parameter distances, 0.0144) between EB and EBBH, (0.0185) between EB and EBOH were greater than average divergence (0.01) between EBBH and EBOH, which suggested that the diverseness between EB and EBBH and EBOH were more than that of between EBBH and EBOH.

Phylogenetic Relationship
Based on the ITS sequences of the three species determined in thisent study and two sequences obtained from GenBank (EB.JS, EF432817; EB.JL, EF432818), parsimony analysis produced two most parsimonious trees, Tree length = 153, with a Consistency index (CI) = 0.9673, Homoplasy index (HI) = 0.0327, CI excluding uninformative characters = 0.9231, HI excluding uninformative characters = 0.0769, Retention index (RI) = 0.9664 and Rescaled consistency index (RC) = 0.9349. As shown in Fig 1, bootstrap 50% majority-rule consensus tree divided the 16 Fructus Evodiae species into three main groups. The three EBOHs, three EBBHs and one EB species formed a biggest group with 54 bootstrap, which include two subgroups, EBOH.ZY1 and EBOH.TR3 with 65 bootstrap, EBBH.TR3 and EBBH.TR2 with 62 bootstrap; the second biggest group with 97 bootstrap comprised of two EBBHs, two EBs and one EBOH species, two EBs species, EB.JS and EB.JL (76 bootstrap)

Contents of Evodiamine and Rutavarpine from Fructus Evodiae
Evodiamine and rutavarpine, two of the main bioactive constituents in Fructus Evodiae, were selected as the reference standards for equalizing and evaluating the quality of the species (Dan et al., 2008). In our experimental chromatographic conditions, the retention time and peak area of Evodiamine were stable and reproducible. Therefore, Evodiamine was selected as the reference peak and marker for Fructus Evodiae in this study. The content of Evodiamine and Rutaecarpine in ten samples was also tested, according to the method of Committee for the Pharmacopoeia of P. R. China (Chi, 2000). The result is shown in the last column of Table 1. Dan et al., (2008) and Poon et al., (2007 ) tested the sequence of ITS of Fructus Evodiae samples from other districts, but GC contents,variation sequence, and in-traspecific sequence divergence in ITS1, ITS1 and 5.8S regions of the entire ITS of Fructus Evodiae in their study were relatively few. Dan et al ., (2008) (30) reported that there was an ITS base sequence variation in length and similarity among different district Fructus Evodiae. The intraspecies identification of the three Fructus Evodiae species from Guizhou, Jiangsu and Shandong province in China was done in this study. When comparing to the former research results, the number of isolates was relatively high and the geography distribution was relatively narrow, which could contribute to the origin and evolution of Fructus Evodiae in Guizhou province of China. According to the ITS sequences of Fructus Evodiae, the results showed that there were variation sites on the rDNA ITS sequence in different isolates, and there was a genetic differentiation in various degrees. Therefore, it is concluded that the ITS sequence of Fructus Evodiae was very conservative. There was an ITS sequence variation among different Fructus Evodiae isolates, which showed that there was a tiny variation among Fructus Evodiae individuals of the same intraspecies.

Discussion
The phylogenetic relationship of Fructus Evodiae isolates from Guizhou, Jiangsu and Shangdong was examined (Fig 1). In this phylogenetic tree, any clade almost includes three species of Fructus Evodiae, i.e. The different kinds  (in the last column of table 1), it is hardly possible to draw a similar conclusion which was deduced by Dan et al., (2008). The reason might be that Dan et al., (2008)'s conclusion is available to relatively wide samplings in geographic areas.

Conclusion
There are two conclusions may be drawn from this study. Firstly, in a relatively-narrow geographic area, the ITS sequence of Fructus Evodiae was very conservative, and there was no ITS sequence variation among different Fructus Evodiae species, therefore, ITS sequence analysis is considered as a non-effective measure for studying the phylogenetic evolution. Secondly, it is difficult to identify and distinguished the three Fructus Evodiae species in detail by using the ITS sequence analysis combined with High Performance Liquid Chromatography (HPLC), therefore a novel test method should be developed to differentiate the three Fructus Evodiae species.