Analyses of Chloroplast Genomic and Morphological Evolutionomy of Yulania Sprengeri and Two New Confusable Species (Magnoliaceae)

To scientifically solve the taxonomical confusion of Yulania sprengeri (Pamp.) D. L. Fu in the world, the chloroplast genomic sequences of four confusable species were assembled and compared with the other species of Yulania Spach by means of the typical algorithm. The results indicated that the evolutionary system of Yulania Spach includes four natural sections: Y. sect. Yulania, Y. sect. Buergeria (Sieb. & Zucc.) D. L. Fu, Y. sect. Rosula D. L. Fu, sect., nov., and Y. sect. Tulipastrum (Spach) D. L. Fu, which all have the same boundaries: PHS (17bp) = 0.96. The species Y. sprengeri (Pamp.) D. L. Fu belong to sect. Yulania, but several confusable species such as Y. diva (Stapf. ex Dandy) D. L. Fu, sp. transl. nov., Y. urceolata D. L. Fu, B. H. Xiong et X. Chen, sp. segregat. nov., Y. pendula D. L. Fu, sp. nov. and Y. viridula D. L. Fu, T. B. Zhao et G. H. Tian belong to the new section Rosula D. L. Fu. According to International Code of Botanical Nomenclature, Y. sprengeri (Pamp.) D. L. Fu is proposed for conservation and emended with the conserved type (D. L. Fu 2017100803, CAF). The misidentified epitype of Magnolia sprengeri, the illustration in Flora of Trees of China, differing taxonomically, selected as Lectotype, with almost equal specimen, paratype (D. L. Fu 2017093001, CAF) from Weining County, Guizhou province of China, was segregated as a new species, Y. urceolata D. L. Fu, B. H. Xiong et X. Chen. The other new confusable species, Y. pendula D. L. Fu, is described and illustrated based on the holotype (D. L. Fu 2012040201, CAF) from Sichuan province of China, which was once misidentified as Y. biondii (Pamp.) D. L. Fu or Y. sprengeri (Pamp.) D. L. Fu. The typci-evolutionary characters including diagnostic differences and particularity of the new section and two new species, are given respectively. The evolutionary system scientifically overcomes the partiality and subjectivity of past taxonomical systems of Yulania Spach of Magnoliaceae Juss.


Introduction
Yulania Spach [1][2][3][4][5][6][7][8][9] is the most primitive taxa of Fructophyta D. L. Fu & H. Fu [10], has a very important position and will play an important role in the evolutionomy of fruit plants. The genus is also difficult taxon and its resources Da- Li Fu et al.: Analyses of Chloroplast Genomic and Morphological Evolutionomy of Yulania sprengeri and Two New Confusable Species (Magnoliaceae) have not yet been ascertained, for being tall trees, growing in the mountains with inconvenient transportation and having more cross-characters and profuse variations of most species, and some of them being very rare in number and very difficult to find. Of course, the slow progress of taxonomy of Yulania Spach is also related to the partiality and subjectivity of traditional plant taxonomy and modern phylogenetic theory. One example is the confusion of Y. sprengeri (Pamp.) D. L. Fu.
The species was published as Magnolia sprengeri Pamp. in Nouv. Giorn. Bot. Ital. in 1915 by R. Pampanini, a Italian botanist, based on the types collected from Mountain Wudang of Hubei province of China by P. C. Silvestri in 1912 and 1913 [11]. "there have been considerable nomenclatural and taxonomical difficulties concerning M. sprengeri and its infraspecific division…because the type material of M. sprengeri…consists only of flowering specimens, and it is not possible to determine the original colour of the dried tepals. Furthermore since the flowers of M. sprengeri are precocious, the specimens lack leaves [12]" But actually the original colour of tepals was not significant, some description of the species was neglected such as the gemma, branchlet and that R. Pampanini had confirmed the species was close to Y. salicifolia (Sieb. & Zucc.) D. L. Fu [1,7,9].
Almost being identical to the illustration of the epitype of Y. sprengeri, a specimen was collected from Weining County of Guizhou province of China at alt. 2300 m in 2017 by D. L. Fu. For its foliar buds glabrous or pubescent, floral (mixed) buds villous differing from "gemmis foliiferis glabris vel ad apicem vix puberulis…alabastris longe sericeo-villosis" of Y. sprengeri Pamp. The suspicion that the illustration differ taxonomically should be scientifically verified. So four chloroplast genomic sequences of Y. sprengeri and three confusable species were assembled, which were compared with the other species of Yulania Spach by means of the typical algorithm based on the evolutionary continuity principle [10]. The results are as follows.

Chloroplast Genome Sequencing, Assembly and Annotation
The leaves of four species of Yulania Spach were collected: Y. sprengeri (Pamp.) D. L. Fu [18]. Using the Illumina Hiseq 2500 platform (Lemont, IL, USA), the high-throughput sequencing was performed after quantification and qualification, and the paired-end library being constructed. All remaining high quality sequences were assembled into contigs using de novo assembly after cleaning the raw data. Using the software SPAdes v3.9.0., the complete chloroplast genomes were assembled. The fully annotated genomes with circular map were drawn in OGDRAWv1.2 [19]. All four chloroplast genomes of Yulania Spach were deposited in the GenBank database.

Evolutionary Analyses of Chloroplast Genomes
Using evolutionary continuity principle of Evolutionomy [10], the morphological characters and the chloroplast genomes of four species of Yulania Spach were compared with other species of the genus. The typical algorithm is mainly adopted in the evolutionary analyses of chloroplast genomes, by comparing the phylogenetic similarity (PHS) between the designated type and objective species or taxon which can determine the relatively evolutionary relationships among different taxa. The formula is as follows: = SPHL APHL PHS = phylogenetic similarity between the type and objective taxon; SPHL = the number of same phylogenetic loci between the type and objective taxon; APHL = all number of phylogenetic loci of the type; statistics of phylogenetic loci using Nucleotide Barcodes (NB≥17bp).

Genomes of Four Species of Yulania Spach
The chloroplast genomes of the four species of Yulania, similar to the chloroplast genomes of other species, include four parts: two inverted repeats, IRA and IRB, and the remaining LSC large single copy area and SSC small single copy area. The complete chloroplast genomes of Y. sprengeri, Y. urceolata, Y. pendula and Y. viridula are 160,096bp, 160,053bp 160,058bp and 160,060bp in length, respectively, which all are encoded 128 genes, include 83 protein coding genes, 8 rRNA genes and 37 tRNAs. atpF, rpoC1, trnL-UAA, trnV-UAC, petB, petD, rpl16, rpl2, ndhB, trnI-GAU, trnA-UGC, ndhA, trnK-UUU, rps16, and trnG-UCC genes with an intron for each gene, clpP and ycf3 gene with two introns, and the rps12 is trans-splicing gene, see Figure 1

Evolutionary System of Yulania Spach
Although the genus Yulania had successfully been established in 1839 by E. Spach, the other genus Tulipastrum Spach, really being same, was also done at the same time by him. Moreover another genus Buergeria was done by P. F. v. Siebold & J. G. Zuccarini in 1845. All these genera usually regarded as Magnolia L. The genus finally had become a natural genus by D. L. Fu [1] based on the results of morphology and DNA phylogeny.
Traditional taxonomical and phylogenic system sometimes are inevitably partial and subjective [10]. To establish a scientific system of the genus Yulania, the PHSs among different species were analyzed based on the chloroplast complete genomes of 14 species of the genus using the types of 4 species respectively: Y.  Table 1 to 4.  It can be seen from Table 1  Deciduous trees and shrubs. Branchlets often greenish, rare purplish (Y. stellata). Floral (mixed) buds often small to 3 cm long. Leaves elliptic to obovate, lower surfaces pubescent, villous or tomentose. Flowers precocious, often lageniform before spreading and tepals often less than 3 cm wide and exconvoluted after spreading. Fruit aggregates ovoid to cylindric. Two New Confusable Species The species of this section are mainly distributed in China, Japan and Korea.
The species of this section are mainly cultivated as a main species for Xinyi, a Chinese traditional medicine.
The new section is easily confused with sect. Yulania, but its particularity such as branchlets often yellow marked with horizontal and horse-shoe-like petiolescars and flowers lotiform, which can be distinguished to the species of Y. sect. Yulania often with reddish purple branchlets with triangular ±protrudent petiolescars or puberulent leaves, and cup-shaped flowers often with less than 12 petals and sepals.
The species of this section are distributed in North America. A good species cultivated for garden or landscaping.

Yulania Sprengeri and Two New
Confusable Species  The chloroplast complete genome of this species is mostly similar to Y. cylindrica (Wils.) D. L. Fu that has known (PHS = 0.993, see Table 1).
The main typici-evolutionary characters are that the hornotini-branchlets yellowish-green rare tannish-green, the leaves obovate, apices usually acumens and lower surfaces pale green and sparsely puberulent, and the tepals 10-12, subsimilar.
It can be easily distinguished from the confusable species of Y. sect. Rosula D. L. Fu such as Y. diva, Y. urceolata, Y. pendula, Y. viridula, Y. sargentiana, Y. dawsoniana and Y. campbellii for its leaf puberulent but that of the confusable species pubescent or villous.
The species, a colloquial name Yingchunhua, is mainly distributed in deciduous broad-leaved forests at an altitude of 600~1700 m in Mountains Qinling and Mountains Daba of China, such as Mountain Baiyunshan in Luoyang, Wuduoshan and Baotianman in Nanyang of Henan province and Mountain Wudang of Shiyan, Shennongjia Forest Region, and Yuan'an county of Yichang of Hubei province, and Ningshan County of Shanxi province. D. L. Fu, B. H. Xiong et X. Chen, sp. Segregat. Nov., Figure 4 Magnolia  The particularity of the new species: foliar buds glabrous or sparsely pubescent, floral buds urceolate or ellipsoideo-urceolate; fruit aggregates red and brown-purple when dried, and stamen scars on fruit receptacle obviously protuberant as thin verrucae, seeds widely nephroid, the width twice as length sometimes.
The species is distributed in the west of Guizhou, northeast of Yunnan, southwest of Hubei, and east of Sichuan province of China at an altitude of 1800~2400 m, colloquial names Hanlianhua, Eryuehua and Furonghua, for its beautifully red or pink flowers. Its floral buds are hard and brushlike, called woodenbrush in ancient China, and also called Xinyi, a Chinese traditional medicine.

Yulania Pendula
The main typici-evolutionary characters of the species: the branchlets stout purplish-red and longitudinally fissured, hornotini-branchlets green or yellowish-green, floral buds large ellipsoid and densely long lanose, the leaves narrow elliptical and lower surfaces pale green, pubescent or villous at least at axils, the stipules linear and pendulous, and the tepals 12~15, red, subsimilar.
The new typici-evolutionary characters of the new species: the foliar buds long columned obviously higher than the gynoecia and the stipules linear and pendulous before falling, which can be distinguished to all other species of genus Yulania Spach.
The species is distributed in northeast of Sichuan province in deciduous broad-leaved secondary forest at an altitude of 1200~2000 m, and cultivated as a main species for Xinyi, a Chinese traditional medicine. So, it is named "Chuan Xinyi" in Chinese for that it was misidentified as Y. biondii (Pamp.) D. L. Fu and Y. sprengeri (Pamp.) D. L. Fu for many years in some botanical authorities and it was really mainly cultivated for "Xinyi" in North Sichuan Province.