Two PEL of cpDNA of Magnolia Accisa, a New Species (Magnoliaceae) from China

Magnolia sect. Rytidospermum Spach is an important taxon and has an important position in the evolutionary and taxonomic system of Magnoliaceae. A new species of the section was discovered in the investigation of the plant resources in China, named Magnolia accisa D. L. Fu, H. Fu et R. M. Duan, sp. nov. for its leaves with accisus apices and viridescent lower surfaces, which had been confused with M. officinalis var. biloba Rehd. & Wils. for many years. In order to determine its evolutionary taxonomic status, two similar species, M. officinalis Rehd. & Wils. with the variety M. officinalis var. biloba Rehd. & Wils, and M. rostrata W. W. Smith were also collected, and two partial sequences of chloroplast genome, matK and trnL-ndhJ, were amplified and sequenced respectively. The partial sequences were analyzed and compared with 39 samples of the chloroplast complete genomes of Magnoliaceae in the NCBI (National Center for Biotechnology Information, USA) database, and the results indicated that the new species Magnolia accisa has two PEL (particularly evolutionary loci): matK (...ACAAGGTGGGATTTCCA...) and trnL-ndhJ (...GTCTAGTCTATAGGAGT...), for that the oppositely evolutionary loci of matK (...ACAAGGTGAGATTTCCA...) and trnL-ndhJ (...GTCTAGTCTAGAGGAGT...) are possessed by all other contrasted samples including the experimental materials and the data of NCBI of Magnoliaceae. So, the new species is described and illustrated according International Code of Botanical Nomenclature, which will speed up the investigation of plant resources of Magnoliasect. Rytidospermum Spach in China.


Introduction
Magnolia sect. Rytidospermum Spach [1][2][3] is an important taxon and has an important position in the evolutionary and taxonomic system of Magnoliaceae, which was even regarded as a new genus, Houpoea N. H. Xia & C. Y. Wu [4,5]. The species of the section had been cultivated more than 2,000 years in China, because its barks has been used as a Chinese medicine, commonly known as Houpo, a traditional Chinese herbal medicines and traditional Chinese exported medicines. The trees grow rapidly, adapt strongly, distribute and are cultivated widely, the flowers are big and fragrant, and the leaves gather at the end of perennial often non-divaricating branches as "umbrella tree" called in North America, so they have unique ornamental features and occupy an important position in the cultivation of Forestry. The species of Magnolia sect. Rytidospermum Spach native to China are mainly three species described: M. officinalis Rehd. & Wils. [6], M. rostrata W. W. Smith [1][2][3][4]7], and M. chathayana D. L. Fu et T. B. Zhao [8]. In the investigation of the plant resources of Magnolia sect. Rytidospermum in Henan, Hunan, Hubei, Sichuan, Yunnan, Xizang and other provinces in recent years, a new species, named Magnolia accisa D. L. Fu, H. Fu et R. M. Duan, sp. nov., had been found by the authors. The new species has the particularity of the leaves with accisus apices and viridescent lower surfaces, but it had been confused with M. officinalis var. biloba Rehd. & Wils. [6,7,9] for many years by botanists [3,[10][11][12]. In order to determine its evolutionary taxonomic status, along with two similar species, M. officinalis Rehd. & Wils. with its variety M. officinalis var. biloba Rehd. & Wils., and M. rostrata W. W. Smith were collected, and two partial sequences of chloroplast genome, matK and trnL-ndhJ, were amplified and sequenced respectively. The partial sequences were analyzed and compared with 39 samples of the chloroplast complete genomes of Magnoliaceae in NCBI (National Center for Biotechnology Information, USA) database, and the results are as follows.  Table 1).

PCR Primer Design
Two pairs of primers, matK-Y01-F and matK-Y01-R, and trnL-ndhJ-Y01-F and trnL-ndhJ-Y01-R were designed using Primer Premier 6, the sequences of primers and the length of amplification and sequencing of cpDNA, see Table 2.

PCR Amplification
Total genomic DNA was isolated from silica-dried leaves of 5 materials belong to 3 species and 1 variety of Magnolia sect. Rytidospermum using a modified CTAB method [13]. The primers of PCR amplification are matK_Y01 and trnL-ndhJ_Y01 (see Table 2) respectively. All PCR reactions were completed in T-gradient (Biometra). PCR amplifications were performed in 15 µL volume: 7.5 µL 2x Es Taq MasterMix, 0.2 µL forward primer and 0.2 µL reverse primer, 6.1 µL ddH 2 O, and 1µL genomic DNA, with the following cycles: 5 min initial denaturation at 94°C, 10 cycles of 30 s at 94°C, 45 s at 61°C and 2 min at 72°C, 27 cycles of 30 s at 94°C, 45 s at 56°C and 2 min at 72°C, and final extension 5 min at 72°C. The PCR products were extracted and purified using the Gel Extraction Kit (OMEGA).

DNA Sequence Analysis
The absolutely coincident partial sequences using the forward primer and reverse primer were correct ones of the samples, which could be analyzed. The particularly evolutionary loci could be easily found out and verified using Microsoft Word or other software. Table 3 listed the contrasted chloroplast complete genomes of Magnoliaceae in the NCBI database.

Two PEL (Particularly Evolutionary
Loci) of cpDNA of Magnolia Accisa

PEL of Partial Sequence of matK of the New Species
There are 706 bp absolutely coincident sequences of Magnolia accisa, using the forward primer and reverse primer of matK-Y01 respectively, which can be regarded as the correct partial cpDNA sequence of the new species. The sequence is as follow: Magnolia accisa, partial sequence of matK of cpDNA ACTTTATTCGATACAAAGTCTGTTTTTTTGAGGAT  CCACTGTGATAATGAGAAAGATTTCTGTATATCCGC  CCAAATCGATTGATAATATCAGAATCTGACGAATCG  GCCCGGACCGACTTACTAATGGGATGCCCTGATACG  TTACAAAATTTCGCTTTAGCCACTGATCCAATCAGA  GGAATAATTGGGACTAGGGTCTCGAATTTATTAATA  GAAGTATCTATTAGAAATGAATTCTCTAGCATTTGA  ATCCTTACCACCGAAGTGTTTAGTCGTACACTTGAA  AGATAGCCCAGAAAATATAAGGAATGATTGTATAA  TTGGTTTATATGGATCCTGTCCGGTAGAGACCACAA  GTAAAAATGACATTGCCAAAAATGGACAAGGTGGG  ATTTCCATTTCTTCATCAGAAGATGAGTCCCCTTTGA  AGCCAGAATGGATTTTCCTTGATATCTGACATAATG  CATGAAAGGGTCCTTGAACAACCATAGGGTCTTCTG  AAAATCATTACGAAGCACTACTACAAGATGTTCTAT  TTTTCCATAGAAATGTGTTCGCTCAAGAAAAGTTCC  AGAGGATGTTGATAGTAAATGAGAAGATTGTTTACG  GAGAAACACTAATACGGATTCACATTCATATACATG  AGAATTATATAGTAACAAGAAGAATCTTTGATTCTC   TTTTGAAAAAAGAGAAATGGATTT  Compared to the

Conclusion
The new species Magnolia accisa has at least two PEL of cpDNA, which are matK (...ACAAGGTGGGATTTCCA...) and trnL-ndhJ (...GTCTAGTCTATAGGAGT...). The leaves with accisus apices and viridescent lower surfaces are also particular in Magnolia sect. Rytidospermum. So the new species is supported by genomic and morphological evolutionomy based on the evolutionary continuity principle and the evolutionary particularity principle. The description of the new species will speed up the investigation of plant resources of Magnolia sect. Rytidospermum Spach in China.