Manglietia pubipedunculata (Magnoliaceae), a new species from Yunnan, China

A new species, Manglietia pubipedunculata Q. W. Zeng & X. M. Hu (Magnoliaceae) is described and illustrated from Yunnan, China. In addition to macromorphological examination, we comparatively studied on micromorphology of leaf epidermis, leaf structure, and epidermal cell on the sclerotesta. This new species is similar to M. kwangtungensis in terms of having dense pubescence, however, their pubescence are quite different. Manglietia pubipedunculata has appressed, compressed, shorter and sparser pubescence consisting of single or two cells. Moreover, it differs from M. kwangtungensis by showing shorter and thicker peduncles, longer styles, basal carpels covered with sparsely brown appressed pubescence, and more ovules per carpel. Furthermore, the new species has thinner leaves, brown and rugged surfaces on sclerotesta, and the alveolate cell pattern consisting of pentagon or hexagon cells with papilla on secondary cell wall under the observation by SEM. The phylogenetic analysis from two nuclear PHYA and LEAFY and chloroplast trnH-psbA sequences of 11 taxa reveals that M. pubipedunculata is a distinct species.


Introduction
China is one of the countries with the highest number of species in the Magnoliaceae throughout the world. More than 100 species of Magnoliaceae are found in China [1]. Southwest and South China including Yunnan, Guangxi, Guangdong, Hainan, Guizhou and the neighbouring areas are considered to be the center of modern distribution and diversity of Magnoliaceae in the world [1][2][3][4].
The genus Manglietia was proposed by Blume [5], but its systematic status has been long debated by taxonomists, some suggested that Manglietia should be reduced to Magnolia [6-a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Sclerotesta morphology
The fresh mature seeds were soaked in warm water with a little washing powder for two days, cleaned with hands to remove the exotesta and mesotesta, and dried naturally, then observed, measured and photographed directly under SV11 ZEISS stereomicroscope. Voucher specimens of both plants from which the seeds originated (Q. W. Zeng & X. M. Hu 00240, Q. W. Zeng & X. M. Hu 00256) were placed in IBSC. The terminology follows Tiffney [46] and Xu [27].

Sclerotesta epidermal cell
After removing the exotesta and mesotesta, the fresh mature seeds were put in a solution of 1:1 xylene and acetone, cooked continuously under 70˚C thermostat metal bath (JS-400A) for one week, and washed at least 3 times with a new solution of 1:1 xylene and acetone in the ultrasonic cleaning machine (1510E-MT) for 30 minutes. Then, the seeds were cleaned with a small amount of 100% of ethanol and aired in a fume hood. Finally, the seeds were mounted on the surface of brass stubs with double-sided tape, and coated with palladium gold using a SPI-MODLE sputter coater. Characters were observed under the JEOL JSM-6360 LV scanning electron microscope operating at 25 kv, and were measured by imaging analyzer (Smile View 2.1; JEOL Tokyo, Japan). Voucher specimens of both plants from which the seeds originated (Q. W. Zeng & X. M. Hu 00240, Q. W. Zeng & X. M. Hu 00256) were placed in IBSC. The terminology follows Karcz et al. [47].
We sequenced three samples of M. pubipedunculata and two samples of M. kwangtungensis for this work. In addition, we downloaded sequences for 9 species of Magnoliaceae from Gen-Bank. GenBank accession numbers for all the DNA sequences and voucher information are given in S1 Table.

Molecular markers
Total cellular DNA was isolated from silica-dried plant leaves using the modified CTAB method [48]. Two nuclear genes (PHYA and LEAFY) and chloroplast gene (trnH-psbA) were analyzed. The primers for PHYA (PHYA-1) and LEAFY (LFY-3) (S2 Table) were modified from those used in Nie et al. [49] based on the sequences of M. moto (EU 849973.1) and M. insignis (EU849837.1), respectively. The trnH-psbA gene region was amplified using the primers as described by Sang et al. [50]. PCR product was sequenced by DNA Analyzer (Applied Biosystems 3730xl).

Sequence alignment and phylogenetic analysis
We aligned the sequences using Clustal W [51], followed by manual adjustments in Bioedit [52]. Test of substitution saturation showed sequences can be used to build phylogenetic tree for Iss.c (0.7971) > Iss (0.0292), and Prob (Two-tailed) was 0.000. Test for homoplasy using PAUP � version 4.0a [53] showed three genes in this study can be combined to analysis (P = 0.1>0.05). Phylogenetic analyses were carried out using Maximum Parimony (MP) and Maximum Likelihood (ML) as implemented in PAUP � version 4.0 [53] and RAxML-7.0.3 [54]. The MP analysis was performed using heuristic searches with 1000 bootstrap (BS) replicates, the ML analysis used the default GTRCAT_GAMMA nucleotide substitution model. Bootstrap support for ML topologies was inferred using the fast bootstrap algorithm with 1000 replicates. Michelia cavaleriei was used as outgroup because it belongs to Magnoliaceae, but was not the species of Manglietia.

Nomenclature
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Micromorphology characteristics of leaf epidermis
The adaxial epidermis of two species were glabrous (Fig 3A and 3G), epidermal cells seemed irregularly in outline (Fig 3A and 3G). There were lots of pubescence (Fig 3B, 3C, 3H and 3I) growing on round-table-shape placentas (Fig 3D and 3J) and stomatal apparatus (Fig 3E, 3F, 3K and 3L) exsiting on the abaxial epidermis. For M. pubipedunculata, the epidermal cells were a bit blurred in outline (Fig 3A), the pubescence was appressed, compressed, and consisted of single or two cells (Fig 3B and 3C), with the average length of 364.2 μm. It covered 40 pubescence per mm 2 . The stomatal apparatus (Fig 3E and 3F) was protrudent slightly and elliptical with the size of 26.9 × 22.4 μm. It had 274 stomas per mm 2 . For M. kwangtungensis, the pubescence was cocked, spiral, and consisted of three cells (Fig 3H and 3I), with the average length of 813.3 μm. It covered 12 pubescence per mm 2 . The stomatal apparatus (Fig 3K  and 3L) was protrudent apparently and elliptical with the size of 29.8 × 22.7 μm. It had 309 stomas per mm 2 .

Micromorphology characteristics of leaf structure
The thickness of leaves were different, the average of M. pubipedunculata was 217.3 μm, with upper epidermis of 29.1 μm, palisade tissue of 62.6 μm, and spongy tissue of 105.9 μm, while  (Fig 4A, 4C, 4D and 4F), and oil cells were distributed randomly in mesophyll in these two species (Fig 4B-4F).

Sclerotesta morphology
Under the stereomicroscope, the chalazal of the two species was pore, which was round and occurred on the upper ventral face (Fig 5B and 5D). The colour of M. pubipedunculata was brown, the shape was oblong in outline (Fig 5A), the size was 6.09 × 5.9 mm. The raphal sinus was broad and deep ( Fig 5B). The sculpture of surface was rugged (Fig 5A and 5B). However, the colour of M. kwangtungensis was black, the shape was bean-like or cordate in outline ( Fig  5C and 5D), the size was 4.07 × 6.54 mm. The raphal sinus was shallow (Fig 5D). The sculpture of surface was corrugated (Fig 5C and 5D).

Characteristics of sclerotesta epidermal cell
The two species had quite different characters under SEM, even though they had the same type of anticlinal cell wall boundary, which was convex linear. For M. pubipedunculata, the cell pattern on the surface of sclerotesta was alveolate consisting of pentagon or hexagon cells (Fig 6A  and 6B), the average cell size was 33.5 × 14.7 μm, the thickness of outer periclinal cell wall was 3.2 μm, and there were papillate sculpture on secondary periclinal cell wall (Fig 6B). For M. kwangtungensis, the cell pattern on the surface of sclerotesta was mainly reticulate consisting of rectangular cells (Fig 6C and 6D), the average cell size was 35.7 × 12.8 μm, the thickness of outer periclinal cell wall was 2.2 μm, and secondary periclinal cell wall was smooth (Fig 6D).

Phylogenetic position
The

Discussion
In our phylogenetic study, the new species, M. conifera and M. kwangtungensis form a clade, which shows the three species have relatively close relationship, which is consistent with morphological characteristics for having pendulous peduncles in the genus [43]. Compared to the glabrous peduncles and bracts of M. conifera, the new species M. pubipedunculata is more similar to M. kwangtungensis for having dense pubescence on peduncles and bracts, this is why we use these two species to make morphological comparative study. Studies on leaf morphology were very important for the specimens examination because lots of specimens had no flowers and fruits [55]. Previous studies on the Magnoliaceae [18-20, 45, 56-61] showed the leaf characters have the taxonomic significance. Leaves may affect by the environment, but the affection just happened on the quantitative characters, the qualitative characters kept relatively stable [62,63]. In our study, both the quantitative characters including the density and length of the pubescence, the leaf thickness, and the qualitative characters such as the hair type, are remarkably different.
For seed plants, the seed characters are of hereditary stability [64]. Researches demonstrated the type of chalazal region, the colour, shape, size, sculpture of surface and epidermal cell of sclerotesta had the taxonomic significance in the family, genera, and species [26,27,46,[64][65][66]. In the present study, notable differences were definitely found between M. pubipedunculata and M. kwangtungensis.