Morphological characteristics and identification
Inner Mongolia Arnebia Radix (Fig. 1), herbs pubescent. Leaves simple, alternate. Inflorescences scorpioid cymes, bracts present. Flowers bisexual, actinomorphic, Calyx usually 5-persistent, Corolla tubular, limb usually 5-parted. Taproot, containing purple matter. According to Flora of China[40] and Flora Reipublicae Popularis Sinicae[41], it was a preliminary judgment that Inner Mongolia Arnebia Radix belong to the family of Boraginaceae.
Inner Mongolia Arnebia Radix, Ovary 4-lobed, style gynobasic (between ovary lobes) (Fig. 2A). Ovules 4, potentially maturing into 4 nutlets (Fig. 2C). Corolla actinomorphic, lobes subequal, Corolla throat unappendaged(Fig. 3A, Fig. 3B). Stamens in single whorl, included, inserted at same level in corolla tube, connective of anthers not subulate, not spirally twisted (Fig. 3A, Fig. 3C, Fig. 3E). Stigma 2-divided(Fig. 3D). According to the identification key to genera of Boraginaceae[40,41], the morphological characteristics of Inner Mongolia Arnebia Radix match up with the description of Arnebia in Flora of China[40] and Flora Reipublicae Popularis Sinicae[41].
Inner Mongolia Arnebia Radix, corolla yellow (Fig. 3A, Fig. 3B), stamens inserted at same level in corolla tube (Fig. 3E), leaf blade less than 1.2 cm wide (Fig. 4). According to identification key to species of Arnebia[40,41], the morphological characteristics of Inner Mongolia Arnebia Radix match up with the description of Arnebia guttata or Arnebia szechenyi in Flora of China[40] and Flora Reipublicae Popularis Sinicae[41]. Summarized the morphological characteristics of Arnebia guttata and Arnebia szechenyi in Table 3 for the further identification of Inner Mongolia Arnebia Radix.
According to Flora of China[23,42], stems of Arnebia guttata usually 2-4, sometimes only 1, erect, and stems of Arnebia szechenyi sparsely branched, 20-30 cm tall. There was no obvious difference between Arnebia guttata and Arnebia szechenyi on the stem morphological characteristics.
In order to further determine Inner Mongolia Arnebia Radix is belong to Arnebia guttata or Arnebia szechenyi, more morphological characteristics comparison has been done between Arnebia guttata and Inner Mongolia Arnebia Radix. Leaves of Arnebia guttata are spatulate-linear to linear, densely hirsute, hairs discoid at base, apex obtuse (Fig. 5D, Fig. 5E). Leaves of Inner Mongolia Arnebia Radix are narrowly ovate to linear-oblong, short strigose and minutely hispid, hairs discoid at base, margin obtusely serrate, apex acute, bristly (Fig. 5A, Fig. 5B, Fig. 5C). Flowers of Arnebia guttata, Inflorescences cymes (Fig. 6A), bracts linear-lanceolate (Fig. 6E). Calyx lobes linear, long hispid (Fig. 6D). Corolla yellow, tubular-campanulate, pubescent outside; limb 7–12 mm wide, lobes broadly ovate or semiorbicular (Fig. 6B, Fig. 6C). Stamens inserted at the middle in corolla tube (Fig. 6C). Ovary 4-lobed (Fig. 6H). Style filiform, apex 2-lobed; stigma reniform (Fig. 6F). Flowers of Inner Mongolia Arnebia Radix, Inflorescences cymes (Fig. 1), bracts same shape as leaves (Fig. 7B, Fig. 5A). Calyx lobes linear, densely hirsute and hirtellous on both sides. (Fig. 7C, Fig. 7E). Corolla yellow, tubular-campanulate, pubescent outside, limb 5–7 mm wide (Fig. 3A, Fig. 7A, Fig. 7D). Stamens inserted at the middle in corolla tube (Fig. 3A). Ovary 4-lobed. Style filiform, apex 2-lobed (Fig. 2A, Fig. 3D). The leveas and flowers morphological characteristics of Inner Mongolia Arnebia Radix match up with the description of Arnebia szechenyi in Flora of China[42].
Nutlets of Inner Mongolia Arnebia Radix, triangular-ovate (Fig. 8A, Fig. 8B), tuberculate (Fig. 8C), short appressed pubescent (Fig. 8D). The nutlets morphological characteristics of Inner Mongolia Arnebia Radix match up with the description of Arnebia szechenyi in Flora of China[42].
Through a comparative study on the relative ubiety between bracts and flowers in inflorescences between the specimen of Arnebia guttata (Fig. 9A) and Inner Mongolia Arnebia Radix (Fig. 9B), we identified the inflorescences morphological characteristics of Inner Mongolia Arnebia Radix had some differences with Arnebia guttata.
The results of DNA sequences data analysis
The success rate of PCR amplification for ITS2 fragments was 100% in our experiments. The lengths of ITS2 sequences for ZC-1 to ZC-5 and ZC-6 to ZC-13 were 224 and 223 bp, respectively.
Phylogenetic tree of 15 sequences was divided into three clades which were identified as Arnebia szechenyi, Arnebia euchroma, and Arnebia guttata, respectively(Fig. 10). ZC-1, ZC-2, ZC-3, ZC-4 and ZC-5 clustered together with EF199863.1, an ITS2 sequence of Arnebia szechenyi which was downloaded from NCBI database that submitted by Hong Kong Baptist University. ZC-6 and ZC-7 clustered together with JX976805.1, an ITS2 sequence of Arnebia guttata which was downloaded from NCBI database that submitted by Xinjiang Institute of Ecology and Geography. The neighbor-joining (NJ) tree also showed that the three species of Arnebia szechenyi, Arnebia euchroma and Arnebia guttata formed three clades individually. This result strongly endorsed the opinion that Inner Mongolia Arnebia Radix should be contained in Arnebia szechenyi, which was consistent with the identification result of Morphological characteristics.
The results of intra-specific distance and inter-specific distance based on genetic divergence were shown in Table 3. The result of genetic variation test based on K2P-distance for ITS2, showed that the minimum inter-specific distance was 15 times higher than the maximum intra-specific distance (inter max = 0.075 vs. intra min = 0.005), indicating that genetic variation of ITS2 demonstrated separately, non-overlapping distributed between intra- and inter-specific samples. Our results showed that the distributions of intra- and inter-specific variation of ITS2 region exhibited distinct gaps. Therefore, Arnebia szechenyi, Arnebia euchroma and Arnebia guttata can be distinguished by ITS2 region. The result approved that the original plant of Inner Mongolia Arnebia Radix was Arnebia szechenyi.