A total of fourteen specimens of three Fructus Evodiae species were collected from Zunyi and Tongren of Guizhou in China, including two vouchers of EB, six species each of EBOH and EBBH, and the sequences of another two species, code EB.JL and EB.JS, were obtained from the Provincial Key Laboratory of Drug Research, Hunan Academy of Traditional Chinese Medicine, Changsha, Hunan Province, 410013 (Dan HUANG et al., 2008). Due to the morphological variation of Petal and leaf of female Fructus 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 Wu 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

An initial alignment of the ITS sequences was generated using CLUSTALW (THOMPSON, J. D et al.,1994; HIGGINS, D. G et al., 1996 ), and then made several minor adjustments manually. The sequences with those published on the ITS of Fructus Evodiae (Dan HUANG et al., 2008; POON Wing-Sem 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, D. L, 2000) on PC with windows platform. Pairwise evolutionary distances between accessions were generated under the Kimura 2-parameter model (Kimura, M , 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.L.Swofford , 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.

The HPLC system consisted of Quat pumps (Model G1311A, Agilent, U.S.A.), a diode array detector (Model G1315B, Agilent, U.S.A.)”. Chromatographic separation was carried out on a Agilent SB C18 column (250mm×4.6mm, 5μm; Elite, China), using a gradient solvent system comprised of (A) CH3CN and (B) H2O(with 0.05% H3PO4 and 0.04% Octane sulfuric acid sodium salt (Chaudhary M. I., 2007). Gradient profile: 0—25 min: linear 95—65% of B; 25—40min: linear 65—60% of B; 40— 55min: linear 60—58% of B; 55—65min: linear 58—25% of B; 65—100min: 10% of B; on-line UV spectra were recorded with detection wavelength of 225nm, column temperature: 25°C. Data were stored on a personal computer.

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 between ITS1 and ITS2, with much less information present in the 5.8S gene (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).

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 sequence 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.

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) were a sister group to EBBH.TR1 with 99 bootstrap. In another two OTUs, EBBH.ZY2 and EBOH.ZY3, form a polyclade with 97 bootstrap; the remaining four species, EBOH.TR1, EBOH.ZY2, EBBH.ZY1 and EB.ZY1 formed a polyclade in this phylogenetic tree as a individual branch.

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 HUANG 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 Zhenguo, 2000). The result is shown in the last column of Table 1.