Phylogenetic Tree of 50 Medicinal Plants in Shandong Province, China

: Medicinal plants are a valuable resource for a variety of products which are important for human needs. The fundamental step in the process has always been the characterization of species. This is the reason which connects the phytopharmaceutical sciences with taxonomy and species identification. The DNA-based method of identification has revolutionized this field in taking the taxonomy to the molecular level. 50 medicinal plants were selected. Two nucleotide sequences of each collected plant were selected and 100 nucleotide sequences downloaded from the NCBI database. MEGA11 Software was used to analyze those nucleotide sequences, and then Maximum Likelihood tree was reconstructed to reveal genetic relationship of 50 medicinal plants. The phylogenetic tree showed that majority nucleotide sequences of 50 medicinal plants can be clustered on the same branch with the same plants of their network.


INTRODUCTION
Medicinal plants are a valuable resource for a variety of products that are important for human needs. With respect to medicinal plants, the use of plant-based materials dates back to ancient civilizations. There are several ancient written records that provide evidence regarding use of plant sources of remedies [1][2]. The knowledge from ancient systems of plant-based remedies has also been used by the modern pharmaceutical industry. There is thus an immense potential for discovery of new drugs from plants based on the ethnomedicinal data [3][4]. About one-third of currently available drugs come from natural products that have a plant origin [5]. Evaluation of these medicinally important chemicals is not an easy task. The fundamental step in the process has always been the characterization of species. This is the reason which connects the phytopharmaceutical sciences with taxonomy and species identification. The DNA-based method of identification has revolutionized this field in taking the taxonomy to the molecular level [6]. There are nearly 11,000 medicinal species in China [7]. In the study, 50 medicinal plants in Shandong Province were selected to explore the genetic relationship by maximum likelihood tree.

METHODS
The medicinal plant species list and therapeutic uses were complied mainly from local pharmacopoeias. Besides the literature review, we conducted an in-depth analysis of the typical therapeutic uses of medicinal plants. According to the Flora of China, species names were standardized. Species-level groups were formed for infraspecific taxa. And then, 50 medicinal plants were collected. A total of 10 categories of medicinal plants were classified according to their traditional use as a remedy for diseases such as musculoskeletal disorder, skin disorder, hepatic disorder and circulatory disorder, etc. The reconstruction of the subfamily level system of the family selects the chloroplast genes pasB, rbcL, matK, ycfl and nuclear gene Xdh that are currently used to establish the high-level taxonomic hierarchy with a relatively slow evolution rate. When establishing a subfamily and genus-level system, the chloroplast genes trnK, trnL-F and nuclear gene ITS with a faster evolution rate were added. When downloading data, try to download gene fragments measured from the same specimen or from the same literature. Collect the screened species names and sequence numbers for the sub-genes to sort out the sampling table, and use the NCBI batch download function to download the original sequence in fasta format according to the sequence number. Two nucleotide sequences of each collected plant were selected and 100 nucleotide sequences downloaded from the NCBI database. In order to ensure that the sequences used are accurate and reliable, according to the maximum likelihood tree, check the suspicious species and analyze the reasons for the suspicious location of the species, further adjust the sampling, replace or delete the sequence, and repeat the screening process until there is no suspicious sequence Appear. MEGA11 Software was used to analyze those nucleotide sequences, and then maximum likelihood tree was reconstructed to reveal genetic relationship of the total of 50 medical plants.

The basic characteristics of the collected 50 MEDICINAL plants
In the study, 50 medicinal plants were collected (Table1). According to the classification of drug action, the collected medicinal plants were divided into 10 groups including Heat-clearing Drug(n=15), Expectorant Cough Suppressant and Anti-asthmatic Drug(n=12), Tonify Deficiency Drug(n=9), Relieving Exterior Disorder Drug(n=4), Discutient Drug, Cathartic Drug(n=3), Dispelling Internal Cold Drug(n=2), Digestant Drug(n=1), Sedative Drug(n=1) and Blood-activating Drug(n=1). Two sequences of each collected plant were selected. And then, 100 sequences downloaded from the NCBI database.   (Figure 4). The result showed that 9 plants cluster into two clades. Rehmannia glutinosa was more closely related with Glycyrrhiza glabra. Atractylodes was more closely related with Eleutherococcus senticosus, Pseudostellaria heterophylla, Angelica, Astragalus complanatus and Cynomorium songaricum. Two sequences of Lycium barbarum were clustered into two clades. The result showed that 4 plants cluster into two clades ((Xanthium strumarium+ Saposhnikovia divaricata)+ Magnolia sprengeri). Two sequences of Ephedra were clustered into two clades.

DISCUSSION
The phylogenetic comparative methods were increasingly applied to analyze medicinal plants data with the main purpose of exploring the potential phylogenetic basis of traditional plant knowledge. In the study, phylogenetic tree was reconstructed to analyze the relationship of the collected medicinal plants in Shandong Province. In this study, we selected 50 medicinal plants because of their huge diversity, the accumulation of long-term experience, and significant preventive/curative effect. Previous studies tested the application of phylogenetics for exploring medicinal uses of plants but were restricted to the flora of specific regions and investigated only one or a few disease categories [8]. Hence, the phylogenetic exploration of medicinal plants at a large scale can help to screen or identify plants with high medicinal value. The collected medicinal plants were divided into 10 groups including Heat-clearing Drug, Expectorant Cough Suppressant and Anti-asthmatic Drug, Tonify Deficiency Drug, Relieving Exterior Disorder Drug, Discutient Drug, Cathartic Drug, Dispelling Internal Cold Drug, Digestant Drug, Sedative Drug and Blood-activating Drug. Maximum Likelihood tree showed that the majority nucleotide sequences of 50 medicinal plants can be clustered on the same branch with the same plants of their network, and can be distinguished from their related plants. Nucleotide sequences of minority medicinal plants were not clustered on the same branch. This may be related to sequencing differences or selected nucleotide sequences.

CONCLUSION
In the study, 50 medicinal plants were selected to analyze relationship. The phylogenetic tree showed that majority nucleotide sequences of 50 medicinal plants can be clustered on the same branch with the same plants of their network.