Analyses of Chloroplast Genomic and Morphological Evolutionomy of Yulania Subsect. Cylindricae (Magnoliaceae)

To scientifically settle the puzzle of origin of fruit plants, the chloroplast genomic sequences of three species of Yulania subsect. Cylindricae (Spongb.) D. L. Fu, subsect. comb. nov. (Magnoliaceae) were determined, which were compared with some taxa by means of the typical algorithm, a new method for genomic evolutionomy based on the evolutionary continuity principle. The results indicated that among some representative species of Gymnospermophyda, Yulania puberula D. L. Fu, sp. nov. has the closest relatively evolutionary relationship with Ginkgo biloba, not with the species of Cycas, Welwitschia or Ephedra, which indicated that fruit plants originated from Ginkgoopsida, not from Cycadopsida thought by the euanthium-theory or Chlamydopsermopsida thought by the pseudoanthium-theory. Among some representative species of Fructophyta, Ginkgo biloba has the closest relatively evolutionary relationship with Yulania puberula indicating that the new species is the relatively most primitive species of fruit plants, which is consistent with the results of morphological evolutionomy. The evolutionary system of Magnoliaceae includes 4 natural genera: Yulania Spach, Magnolia L., Michelia L. and Liriodendron L., whose boundaries all are PHS(17bp)=0.93. Furthermore Yulania subsect. Cylindricae and its three species were described or emended. The holotype of new species of Yulania puberula was designated, whose main typici-evolutionary characters, including diagnostic differences and particularities, was given and illustrated. The epitype of Y. shizhenii was designated and four synonyms of Y. cylindrica were listed. Typical algorithm is a scientific method of genomic evolutionomy and a scientifically new tool to solve the puzzle of evolutionomy of fruit plants.


Chloroplast Genome Sequencing, Assembly and Annotation
Total genomic DNA was isolated from silica-dried leaves of three species of Yulania subsect. Cylindricae: Y. cylindrica (Wils.) D. L. Fu from Xiuning county of Anhui province, Y. shizhenii D. L. Fu et F. W. Li from Chengdu City of Sichuan province and new species of Y. puberula D. L. Fu from Mountain Wudang in Hubei Province, using a modified CTAB method [9]. After quantification and qualification, a paired-end library was constructed, and high-throughput sequencing was performed using the Illumina Hiseq 2500 platform (Lemont, IL, USA). After cleaning the raw data, all remaining high quality sequences were assembled into contigs using de novo assembly, and the complete chloroplast genomes were assembled using the software SPAdes v3.9.0. The circular map of fully annotated genomes were drawn in OGDRAWv1.2 [10]. All three chloroplast genomes of Yulania subsect. Cylindricae were deposited in the GenBank database.

Evolutionary Analyses of Chloroplast Complete Genomes
By analyzing the morphological characters using evolutionary continuity principle of Evolutionomy [1], the chloroplast genomes of three species of Yulania subsect. Cylindricae (Spongb.) D. L. Fu, subsect. comb. nov. (Magnoliaceae) were compared with other primitive groups such as Amborellaceae Pinch., Winteraceae Lindley, Trochodendraceae Prant., in order to scientifically determine the evolutionary position of Yulania subsect. Cylindricae (Spongb.) D. L. Fu in the evolution of fruit plants. The evolutionary analyses of chloroplast genomes mainly adopted the typical algorithm, by comparing EVS = the evolutionary similarities between the designated type and species or taxa, and PHS = the phylogenetic similarity between the designated type and species or taxa, to determine the relatively evolutionary relationship among different taxa. The formulas are as follows: EVS=SEVL/AEVL EVS = evolutionary similarity; SEVL = same evolutionary loci between the type and taxon; AEVL = all evolutionary loci between the type and taxa (evolutionary loci statistics just including SNB of transitions and transversions in the middle of barcode; SNB = Single Nucleotide Barcode) PHS=SPHL/APHL PHS = phylogenetic similarity; SPHL =same phylogenetic loci between the type and taxon; APHL = all phylogenetic loci of the type (the loci statistics according NB; NB =Nucleotide Barcode)

Origin of Fruit Plants
According to the evolutionary continuity principle, it is known that Fructophyta D. L. Fu & H. Fu originated from Gymnospermophyta D. L. Fu & H. Fu [1], but we don't know which taxa of Gymnospermophyta have the closest evolutionary relationships with fruit plants especially about the main evolutionary vein of origin and evolution of fruits. Because the new species Yulania puberula D. L. Fu of subsect. Cylindricae (Spongb.) D. L. Fu has the most primitive peruloid bracts (see Figure 3 (7)), so it is selected as the type of fruit plants and calculated the evolutionary similarity and the phylogenetic similarity among the samples of Gymnospermophyta from NCBI, the results are shown in Table 3.
It can be seen from Table 3 that there are 1,69 1evolutionary loci (SNB = 21bp) between the species of Yulania puberula and gymnosperms, of which 453 are the same with the species of Ginkgo biloba and the EVS is 0.2679, which is higher than other gymnosperm samples. Therefore Yulania puberula and Ginkgo biloba has the closest relatively evolutionary relationship. PHS between Yulania puberula and Ginkgo biloba (PHS=0.0548) is also obviously higher than other samples of gymnosperms. According to the evolutionary continuity principle, it can be determined that fruit plants originated from Ginkgoopsida, not from Cycadopsida thought by the euanthium-theory or Chlamydopsermopsida by the pseudoanthium-theory.

Relatively Evolutionary Relationships Among Different Classes or Subclasses of Fructophyta
For only one species survived in Ginkgoopsida, so Ginkgo biloba is consequentially selected as the type of Gymnospermophyta. To represent 5 classes and 20 subclasses, 22 species were selected and calculated the evolutionary similarity and the phylogenetic similarity with the type, the results are shown in Table 4.
It can be seen from Table 4 that EVS and PHS between Ginkgo biloba and Yulania puberula is 0.2776 and 0.0608 respectively, obvious higher than other species such as Trochodendron aralioides and Amborella trichopoda. So Magnoliidae is primitive subclass of fruit plants. it is not supported that Trochodendron Siebold & Zucc. are original family in H. D. Zhang system and Amborella Baill. Are original family in APG system by chloroplast genomic evolutionomy.

Relatively Evolutionary Relationships Among Different Families of Magnoliidae
For only 10 families having chloroplast complete genomes in NCBI, 10 species were selected and calculated the EVS and PHS with Ginkgo biloba, the results are shown in Table 5.
It can be seen from Table5 that EVS and PHS between Ginkgo biloba and Yulania puberula is 0.2475 and 0.0608 respectively, obvious higher than other species such as Drimys granadensis. So Magnoliaceae is the most primitive family of fruit plants. It is not supported that Winteraceae R. Br. ex Lindl. were the most primitive family in Takhtajan system and Cronquist system by chloroplast genomic evolutionomy.

Relatively Evolutionary Relationships Among Different Genera of Magnoliaceae
To scientifically reflect the relatively evolutionary relationships among different genera of Magnoliaceae, 28 species having chloroplast complete genomes in NCBI were selected and calculated the evolutionary similarity and the phylogenetic similarity with the type of Ginkgo biloba, the results are shown in Table 6.
It can be seen from Table 6 that EVS between Ginkgo biloba and Yulania diva? is 0.0859 obvious higher than other genera and other species such as Magnolia grandiflora, Liriodendron tulipifera and Michelia laevifolia. So it can be concluded that Yulania Spach is the most primitive genus of Magnoliaceae. It is not supported that Magnolia L. is the most primitive genus in Hutchinson system and C. Y. Wu system (Manglietia Blum.=Magnolia L.).

Relatively Evolutionary Relationships Among Species of Genus Yulania
In order to determine the relatively evolutionary relationship among the species of genus of Yulania, the EVS and PHS between Ginkgo biloba and 10 species of Yulania Spach were calculated, and the results are shown in Table 7: It can be seen from Table 7 that Yulania puberula and Yulania cylindrica have the closest relatively evolutionary relationships with Ginkgo biloba, and the evolutionary similarities are same as EVS=0.0255, but PHS between Yulania puberula and Ginkgo biloba is PHS=0.0713, which is higher than PHS= 0.0712 between Yulania cylindrica and Ginkgo biloba, so Yulania puberula has the closest evolutionary relationship and phylogenetic relationship with Gingko biloba among species of the genus of Yulania Spach.
So it can be concluded that Magnoliaceae Juss. are the most primitive family, Yulania Spach is the most primitive genus and Yulania puberula is the most primitive species of Fructophyta.
Traditional taxonomical and phylogenic system sometimes are inevitably partial and subjective [1]. To establish a scientific system, the PHS and EVS are analyzed based on the chloroplast complete genome of 28 species of Magnoliaceae using the types of 4 species respectively: Yulania denudata (Desr.) D. L. Fu, Magnolia fordiana (Oliver) Hu, Michelia odora (Chun) Nooteboom & B. L. Chen, Liriodendron tulipifera L., and the results are shown in Table  8 to 11.
It can be seen from According the evolutionary continuity principle, based on PHS and EVS of 28 species, the evolutionary system of Magnoliaceae Juss. can be established as Figure 2, which scientifically overcomes the partiality and subjectivity of traditional and phylogenic system. The genera of Magnolia L., Michelia L. and Liriodendron L. of Magnoliaceae Juss. were widely accepted by most of taxonomical system [2-8, 11-13, 15-19, 21-23], but the genus of Yulania Spach had been regarded as Magnolia L. by most botanical authorities [16][17][18]. As Yulania Spach was scientifically confirmed in 2001 [19], its species resources were also gradually identified [20][21][22][23] The species of this subsection are the most primitive taxa of fruit plants, and plays an important role in the study of origin and evolution of fruit plants. D. L. Fu, sp. Nov., Figure 3 Arbor decidua. Ramuli    The main typici-evolutionary characters including diagnostic differences and particularities of the new species are that the hornotini-branchlets reddish purple, leaves thin corious, elliptic or oblanceolati-elliptic, reticulate veins depressed and rimous when dried, lower surfaces densely puberulent; Flower precocious, sepals 3 and tepals 6, bract 1, peruloid. That the leaves oblanceolati-elliptic, reticulate veins depressed and rimous when dried, bract peruloid are also the new typci-evolutionary characters of the species, which can be used to distinguish to similar species Yulania cylindrica (Wils.) D. L. Fu [16][17][18][19][20][21][22][23], Y. shizhenii D. L. Fu et W. F. Li [20] and other species of the genus. Furthermore the leaf reticulate veins depressed and rimous when dried and bract peruloid are also the most primitive morphological signs in fruit plants.

Yulania Puberula
Distribution: The species is mainly distributed in Hubei province of China.
The species is the most primitive fruit plants and will play an important role in researching the Evolutionomy of Magnoliaceae Juss. even that of Fructophyta D. L. Fu& H. Fu, but it is very rare in population, so the authors intensively proposed that the species should be protected as first level protection. Characteres specifici peculiares prioritatum et evolutionales typici: Ramuli graciles cum limbo-cicatricibus petiolorum hornotini purpureo-rubri. Folia longe obovata membranacea subtus dense puberula, petiolis base dilatatis. Flores ante folia aperti; sepala 3 et tepala 6; stamina alba, filamentis latiora quam antheris.
Typus  This species is named for commemorating Li Shizhen, a great pharmacologist and botanist of ancient China.
Distribution: The species is distributed in Sichuan province of China. The species is an excellent ornamental tree species with the pure white flowers, open in early spring. Its flower buds into Chinese medicine, called Xinyi. As one of the most primitive taxa, it should be recommended for inclusion in the national first-level list of protected plants for its small number of populations and very important role in the origin and evolution of fruit plants.