The genus Veratrum L. is one of the most important groups in the family Melanthiaceae (Liliflorae), with approximately 17–45 species of perennial herbaceous plants [1–5]. It is widely distributed in the temperate to arctic zones of the Northern Hemisphere, the majority of which are native to Eastern Asia [1, 2, 6]. There are roughly 13 species and one variety of Veratrum plants in China, several species were used in medicine for more than 1700 years, including V. nigrum, V. schindleri, V. maackii and others [4, 5, 7, 8]. For instance, the dried roots and rhizomes of V. nigrum (Li-lu in Chinese) have been used to treat aphasia arising from apoplexy, wind-type dysentery, scabies, jaundice, and chronic malaria [7, 8]. Other species V. taliense, V. stenophyllum, V. mengtzeanum, and V. grandiflorum that are the source of folk medicine “Pimacao” in China, has been used for treating bruises, rheumatic pain and wound hemostasis [8–10]. Furthermore, “Pimacao” has a high economic value and is one of the main components of Yunnan Baiyao, as well as the principal drug of Yilizhitong pill [10, 11]. The major active ingredient isolated from roots of the above Veratrum plants is steroidal alkaloids, which pharmacological activities mainly focus on decreasing blood pressure, anti-platelet aggregation and anti-thrombosis, and have anti-inflammatory, analgesic and antitumor effect [11–14]. However, Veratrum plants also contain toxic components such as cevanine-ester alkaloids [13–15]. In human nausea, bradycardia, hypotension and apnea develop shortly after ingestion, in some cases resulting in death [12, 13, 15]. Our previous investigation found that some closely related species are occasionally mixed with Veratrum medicinal varieties as adulterants undermining the security and efficacy of the Veratrum containing medicinal products. Thus, securing accurate identification is urgent for the utilization of Veratrum medicinal materials safely and effectively.
Veratrum has a controversial taxonomic history and has been combined with Melanthium totally or in part [1, 16–19]. Since Linnaeus (1753) first circumscription of Verarum, many subgenera, sections, and subsections have been proposed [1, 16–27]. The first subgeneric division of Veratrum was suggested by Baker (1880) [21]. Using perianth coloration as a diagnostic trait, he divided the genus into informal groups: stirps V. albi (perianth white-green) and stirps V. nigri (perianth purple-black) [21]. Loesener split the genus Veratrum into three subgenera and two sections including 48 species consisting of elements of Melanthium [22–24]. Nakai modified Loesener's infrageneric classification into two sections, omitting the rank of subgenera, and divided each section into two subsections [25, 26]. Recently, Veratrum has been circumscribed broadly (including Melanthium) and divided into two sections (sect. Veratrum and sect. Fuscoveratrum) and two subsections (subsect. Pseudoanti and subsect. Asiaveratrum) [1]. This modified infrageneric classification established a framework for resolving phylogenetic relationships within Veratrum [1, 6].
Morphologically, the key characteristics of Veratrum vary greatly depending on habitat, environment, and developmental stages, and the range of those variations often overlap among the taxa [1, 22–24, 28]. As a result, some species and its closely related species constitute a taxonomically complex group that is difficult to be clearly distinguished based on morphological traits alone such as V. maackii complex group [1, 6]. V. maackii has been divided into numerous varieties by different authors, including V. japonicum (broader leaves; relatively large flowers), V. schindleri (broader leaves; short-pedicellate flowers) and so on [1, 4, 5, 25, 26, 29–31]. Shimizu pointed that V. japonicum was a variety of V. maackii and was recognized by WCSP (World Check List of Selected Plant Families; http://www.theplantlist.org). In Flora Reipublicae Popularis Sinicae (FRPS), V. japonicum and V. schindleri were treated as two distinct species from V. maackii [4]. In addition, V. japonicum was treated as a synonym of V. schindleri in Flora of China (FOC) [5]. At the same time, because of its similar characters, such as black-purple perianth, basal leaves, and bulb layers disintegrating into reticulated fibers, V. maackii has been historically placed either in the V. nigrum or treated as its subspecies [32].
Veratrum species were a subject of many molecular analyses [1, 2, 6, 33–35]. Kim et al. conducted maximum parsimony and Bayesian inference based on ITS and cpDNA regions (matK, psbA-trnH, rpl16, and trnS-G)to re-examine the taxonomic status and phylogenetic relationships within Veratrum in Korea and Japan [33]. An analysis using the sequences of ITS, trnL-F, and atpB-rbcL indicated that Veratrum possibly originated in East Asia and radiated across the Northern Hemisphere, but most of the species were not well distinguished [6]. Previous studies have significantly advanced the phylogeny and taxonomy of genus Veratrum. However, due to limited resolution of molecular phylogeny and insufficient sampling of Asiatic species, phylogenetic relationships among the species of the main clades (sections and subsections), particularly among the species from East Asia, are still poorly understood [1, 2, 6]. These shortcomings motivated our study.
The chloroplast genome of higher plants is relatively conservative in its structure, being a double-stranded circular molecule of 120–160 kb in length and comprising a large single-copy (LSC) region and a small single-copy (SSC) region, separated by two identical copies of inverted repeats (IRs) regions [34–36]. The cp genome has been widely used for evolutionary, taxonomic and species diversity studies due to such features as highly conserved genome structure, maternal inheritance, low to moderate evolutionary rate and low effective population sizes [36, 37]. The cp genomes have been showed to be effective in resolving problematic phylogenetic relationships at different taxonomic levels [38–41]. Up to now, however, the plastomes of only a few Veratrum species have been sequenced, and the data accumulated are still deficient for the clarification of the internal relationships of the family [35, 42–44]. Hence, we attempted to explore more Veratrum and its related species phylogenetic relationships with chloroplast genomics.
Here, we sequenced, assembled and annotated the complete cp genomes of ten Veratrum species using the next-generation sequencing platform, and performed the first comprehensive analysis of Veatrum species from China. This study aimed to: (1) establish and characterize the newly sequenced plastomes of ten Veratrum species; (2) examine the variations of SSRs and LSRs among these plastomes plus two previously published plastomes of Veratrum; (3) discover the most variable regions that could be used as DNA barcodes for Veratrum; (4) and reconstruct phylogenetic relationships among the Veratrum species using the plastome sequences.