Two Particularly Evolutionary Loci of trnL-ndhJ of cpDNA of Yulania Baotaina, a New Species (Magnoliaceae) from China

The partial sequence of trnL-ndhJ of chloroplast genome ofthe new species of the genus Yulania Spach of Magnoliaceae, Yulania baotaina D. L. Fu, Q. Zhang et M. Xu, collected from Baotai Mountain of Yongping County of Yunnan province of China, and the similar species, Y. campbellii (Hook. f. et Thoms.) D. L. Fu, collected from Yongping and Gongshan county of Yunnan, and Yadong county of Xizang, were amplified and sequenced respectively, which were compared with 39 samples of the chloroplast complete genomes of Magnoliaceae in the NCBI (National Center for Biotechnology Information, USA) database. The results indicated that Y. baotaina has two PEL (particularly evolutionary loci): trnL-ndhJ (...TTTCATTTCGGAATTTCCTATTTTCTT...) not possessed by others, and the oppositely evolutionary loci of transversions trnL-ndhJ (...TTTCATTTCTGAATTTCATATTTTCTT...) is possessed by all contrasted experimental materials and most simples of Magnoliaceae in NCBI. So the new species was described and illustrated. Its floral buds cylindrical, ovaries subcordiform, and peduncles of flower and fruits obviously elongate are particular, which can be easily distinguished to the similar species and all other ones in the genus. The new species is distributed in Hengduan Mountain of west Yunnan province and south Xizang Autonomous Region at an altitude of 1900~3000 m, a colloquial name is Mulianhua, just as the similar species Y. campbellii. The designated type of the new species was collected from Baotai Mountain of Yongping County at altitude of 2600 m and kept at the Chinese Academy of Forestry. This paper will provide a new research method that can be easily verified for the evolutionary taxonomy and the new science of evolutionomy.


Introduction
As the most primitive taxa of Fructophyta D. L. Fu & H. Fu [1], Yulania Spach [2][3][4][5][6][7][8][9][10] has a very important position and plays an important role in the evolutionomy of fruit plants. The large and beautiful early spring flowers of Yulania Spach were also deeply impressed and loved by the world's gardeners. For example, Yulania campbellii (Hook. f. et Thoms.) D. L. Fu (Magnolia campbellii) [2][3][4][5][6][8][9][10][11][12][13][14], native to a wide area of the Himalayan region, such as E Nepal, Bhutan, N India, Burma, and Xizang Autonomous Region and Yunnan province of China, had attracted admirations from horticulturists in the world, just as that D. Hunt had written "A variable species, spectacular in bloom" [11], and that D. J. Callaway had written "This species is often considered by writers and gardeners to be the most spectacular of all magnolias, and the most desirable" [12]. In the investigation of the plant resources of Yulania Spach, a new species, named Yulania baotaina, was found by the authors. It is very similar to the most spectacular and most desirable species, Y. campbellii (Hook. f. et Thoms.) D. L. Fu. In order to determine its evolutionary taxonomic status, the new species and the similar species were collected from Yunnan province and Xizang Autonomous Region of China, and the partial sequences of chloroplast genome, 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 are as follows.

Plant Materials
Seven samples of the leaves of two similar species were collected from China: two of the new species of Yulania baotaina from Mountain Baotai in Yongping County of Yunnan province, five of the similar species Yulania campbellii (Hook. f. et Thoms.) D. L. Fu from Yongping and Gongshan County of Yunnan province and Yadong county of Xizang Autonomous Region respectively (see Table 1).

PCR Primer Design
A pairs of primers, 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 7 materials belong to 2 species, 2 samples of Y. baotaina and 5 of Y. campbellii, using a modified CTAB method [15]. The primers of PCR amplification are trnL-ndhJ_Y01 (see Table  2). PCR amplifications were performed in 15µL volume containing 0.2 µL forward primer and 0.2 µL reverse primer, 1µL genomic DNA, 7.5 µL 2x Es Taq MasterMix, 6.1 µL ddH 2 O, and 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 5 min final extension at 72°C. All PCR reactions were performed in T-gradient (Biometra). The amplified products of PCR were extracted and purified with the Gel Extraction Kit (OMEGA). New Species (Magnoliaceae) from China

DNA Sequencing
The purified DNAs were sequenced using ABI 3730 XL. The PCR amplifications were performed in 15µL volume containing 0.2 µL forward primer and 0.2 µL reverse primer, 1µL purified DNA, 7.5 µL 2x Es Taq MasterMix, 6.1 µL ddH 2 O, and with the following cycles: 3 min initial denaturation at 95°C; and 26 cycles of 10 s at 95°C, 10 s at 50°C and 4 min at 60°C.

DNA Sequence Analysis
The partial correct sequences of absolutely coincident sequencing using the forward primer and reverse primer were analyzed. The particularly evolutionary loci could be easily found out and verified using some software such as Microsoft Word. The contrasted chloroplast complete genomes of Magnoliaceae in the NCBI database see Table 3.

Two PEL (Particularly Evolutionary Loci) of cpDNA of Yulania Baotaina
There are 810 bp absolutely coincident sequences of Yulania baotaina, using the forward primer and reverse primer of trnL-ndhJ_Y01 respectively, which can be regarded as the correct partial cpDNA sequence of the new species. The sequence is as follow: Yulania baotaina, partial sequence of trnL-ndhJ of cpDNA TTCTCATTCACTCTACTCTTTCACAAATGGGTCCGA  CCATAAATGTTTCTCTCTTATCACAAGTCTTGTGATAG  ATATGATATACGTACAAATGCCCATATTATGGGCAAGG  AATCTCCATTATTGAATCATTCACAGTGCATATCATTA  CTCTTACACTTACAAAGCCTTCTTTTTGAAGATCCAA  GAAATTCCAGGACCTAGGTAAGATTTTGGAATGCAA  TTTGAGTCCCTTTAATTGACATAGACCCAAGTCCTCT  AGTAGGATGACGCATCGGGAATGGTCGGGATAGCTC  AGCTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCA  CCAGTTCAAATCTGGTTCCTGACACGTGGTTAATGTA  TCAAACGGATACTCATACAAATGAATCGATATGGATC  GGGATCCATATTTATTAATAGACTAATAGTCTAGACCA  TGATACATAAGTCTAGACCATGATACATACTTATCCAT  CTAGGTATATAAATGGATACCTCCCTTTTTGTAGATGG  GTAAAGAATATATGGGTAAGGTAAAGAAAAGAATTA  GATTCTATTCCCTCTTCTTTTTTGTTTGTTTATACTGTA  CCTCCTCTCGCTCAAAAAGAATGTTAATACTTCATAC  ATATCCGAAGTTAGTTGGCTTGGCTGAAAAACCCAA  AAGTCTAGTCTAGAGGAGTGGAAGGATAGGAATAGA  CAGGATTCATTTCAGATACAATACAAAGAAAGAAAA  TCCGATCCCTTTTCATTTTCATTTCGGAATTTCCTATT  TTCTTTCATATTCTATTTCTTCACTTTCTATT  Compared to its  So, the new species, Yulania baotaina, is supported by cpDNA evolutionomy based on the evolutionary continuity principle [1] and the evolutionary particularity principle, for that it has at least two PEL of cpDNA trnL-ndhJ (...TTTCATTTCGGAATTTCCTATTTTCTT...). According International Code of Botanical Nomenclature [16], the new species should be described and illustrated.  The flowers of the new species are large, purple and colorful, and graceful. The floral buds are large and cylindrical, and the fruit is dark red and has various shapes. It is an excellent ornamental tree species. Floral buds are used in traditional Chinese medicine for "Xinyi" and are also the raw materials for extracting essence.

Conclusion
Yulania baotaina has at least two PEL of cpDNA, which are trnL-ndhJ (...TTTCATTTCGGAATTTCCTATTTTCTT...). Its floral buds cylindrical, ovaries subcordiform, and peduncles of flower and fruits obviously elongate are particular, which can be easily distinguished to the similar species and all other ones in the genus. So the new species is supported by cpDNA evolutionomy and morphological evolutionomy based on the evolutionary continuity principle and the evolutionary particularity principle, which provides a new research method that can be easily verified for the evolutionary taxonomy and the new science of evolutionomy.