Prunus pananensis (Rosaceae), a New Species from Pan'an of Central Zhejiang, China

Prunus pananensis Z. L. Chen, W. J. Chen & X. F. Jin, a new species of Rosaceae from central Zhejiang, China is described and illustrated. Micromorphological characters of the indumentum on young shoots, leaves, petioles and peduncles, including scanning electron microscope [SEM] images, are provided. This new species is morphologically similar to P. schneiderianae Koehne in having its young shoots, petioles and pedicels all densely villose, but differs in having bracts persistent, styles glabrous, stipules 8–9 mm long, stamens 28–30 of per flower, and drupes glabrous. The new species is also similar to P. discoidea (Yü & C. L. Li) Yü & C. L. Li ex Z. Wei & Y. B. Chang in having 2 or 3 flowers in an umbellate inflorescence, and bracts persistent and marginally glandular, but it differs in having young shoots and petioles densely covered with yellowish-brown villose trichomes; leaves rounded or slightly cordate at base, the mid-ribs and lateral veins abaxially densely covered with yellowish-brown villose trichomes; and hypanthium ca. 3 mm long, shorter than sepals. The atpB-rbcL and trnL-F intergenic chloroplast spacers are selected for identification of the new and its similar species.


Introduction
Fruit type, carpel numbers and ovary position are frequently used for the traditional classification of the family Rosaceae. Four subfamilies, Spiraeoideae, Rosoideae, Maloideae and Prunoideae, were proposed. This subfamilial classification of Rosaceae is currently in a state of flux, and most systematists no longer recognize the Prunoideae, which is instead now included within an expanded Spiraeoideae [1]. Spiraeoideae is now replaced by Maloideae.
Subg. Cerasus (Mill.) Focke, comprising ca. 40 species, includes deciduous fruit trees and garden ornamentals and is mainly distributed in eastern Asia [4,6]. Koehne proposed a system of subg. Cerasus worldwide, and two groups (not ranked) with four sections, 14 subsections and several series were established [7]. Although Cerasus is no longer recognized at the generic rank in most floras, this subgenus has been treated as an independent genus with several sections in China. There are 44 species of subg. Cerasus in China, and the independent genus Cerasus was used by Li and Bartholomew [6]. Yü and Li proposed a generic system of the Chinese species, with two subgenera and 11 sections recognized [5,6].
During 2010 to 2012, the senior author (Jin) organized botanical trips in Dapanshan National Natural Reserve and the adjacent regions of Pan'an County, Zhejiang Province, China. In these trips, previously undescribed species of Prunus in sect. Lobopetalum (Koehne) Yü & C. L. Li was collected. This species is morphologically similar to P. schneiderianae Koehne in having young shoots, petioles and pedicels that are all densely villose, but differs in having bracts persistent, styles and drupes glabrous, stipules 8-9 mm long, and stamens 28-30 of per flower. The species is also similar to P. discoidea (Yü & C. L. Li) Yü & C. L. Li ex Z. Wei & Y. B. Chang in having 2 or 3 flowers in umbellate inflorescence, and bracts persistent and glandular at margin, but differs in having young shoots and petioles densely pubescent with yellowish-brown villose trichomes; leaves rounded or slightly cordate at base, their mid-ribs and lateral veins abaxially densely covered with yellowish brown villose trichomes; and hypanthium ca. 3 mm long, shorter than sepals. Based on studies on morphology and DNA barcoding, we concluded that it represents a new species, described below.

Taxon sampling
The new species and its putative relatives, Prunus discoidea and P. schneideriana, were sampled for this study. Prunus serrulata, the species grew together with Prunus discoidea and P. schneideriana in eastern China, was selected as the outgroup. Prunus serrulata also has spreading sepals, petals 2-lobed, drupes purplish black and inflorescences subumbellate, but its bracts are deciduous. Fresh leaves were collected from a total of 16 individuals from different populations in Zhejiang and Anhui (Table 1)

SEM observation
The indumentum on young shoots, leaves, petioles and peduncles of Prunus discoidea, P. schneideriana and P. pananensis was observed using a Philips XL-30E scanning electron microscope (SEM). The sampled materials were cleaned in 50% ethanol for 30 min, and air dried. The cleaned materials were mounted on stubs using double-sided adhesive tape, and sputter-coated with gold prior to SEM observations.

DNA extraction, PCR and sequencing
Samples for DNA extraction were dried in silica gel. Total genomic DNA was extracted using standard CTAB method [12]. The PCR amplifications were carried out on a DNA Engine PCR (Bio-Rad) in 50 mL reactions, and two chloroplast DNA regions (namely atpB-rbcL and trnL-trnF) were used [13]. Each reaction contained 5.0 mL 106buffer, 2.0 mL dNTPs (2 mmol/mL), 1.0 mL each primer (10 mmol/mL), 4.0 mL genomic DNA (20 ng), 0.5 mL Taq polymerase (5 U/mL), and 36.5 mL ddH 2 O. The PCR program began at 94uC for 1 min, followed by 34 cycles of  94uC for 30 s, 50uC for 30 s and 72uC for 2 min, followed by a 72uC extension for 5 min [13]. All PCR products were electrophoresed on 1% agarose gel to verify product size. PCR products were purified with AxyPrep PCR Clean-up Kit (Axygen, China) following the manufacturer's instructions. Sequencing was carried out using PCR primers on an ABI 3730 automated sequencer (AppliedBiosystems, USA).

Data analysis
The non-coding regions have relatively higher evolution rates, as well as more informative characters, and are typically more useful in identifying species than are coding regions [13][14][15]. As recommended by Quan and Zhou, atpB-rbcL and trnL-trnF were selected as DNA barcoding regions for species identification in Prunus. The edited data matrix was analyzed using ClustalX 1.83 to obtain an initial multiple alignment, keeping default alignment parameters [16]. Phylogenetic relationships were analyzed with PAUP* 4.0b10 for maximum parsimony (MP) and MrBayes v.3.0b4 for Bayesian inference (BI), respectively [17,18]. Both MP tree and Bayesian tree were conducted for the concatenated data of atpB-rbcL and trnL-trnF sequences, and gaps were treated as zero (''missing''). For the MP analysis, a heuristic search algorithm with 1000 random addition replicates and tree bisection and reconnection (TBR) branch-swapping. Node support was assessed using 1000 MP bootstrap (BS) replicates. Using Akaike information criterion in Modeltest 3.7, the most appropriate model of sequence evolution for BI was estimated [19]. The analyses were conducted with four Metropolis-coupled Markov chains in each of two independent runs of 10 7 generations. A 50% majority-rule consensus tree was computed by PAUP* after ''burn-in'', and the posterior probabilities (PP) were calculated.

Indumentum on young shoots, petioles, leaves and pedicels
The indumentum on young shoots, petioles, leaves and pedicels is shown in Figure 1. The indumentum on young shoots and petioles of the new species Prunus pananensis and the similar species P. schneideriana is densely villose (Fig. 1: A, B, E, F). The leaves of these two species were villose on costa and lateral veins abaxially ( Fig. 1: C, G), and their pedicels were also villose ( Fig. 1: D, H). In contrast, the young shoots and petioles of P. discoidea were sparsely villose ( Fig. 1: I, J), and the leaves were sparsely villose on costa and lateral veins abaxially ( Fig. 1: K). The pedicels of P. discoidea were glabrous (Fig. 1: L).

DNA barcoding/molecular phylogenetic analysis
The combined atpB-rbcL and trnL-trnF dataset consisted of 17 individuals belonging to four species, and included 1717 aligned characters. The atpB-rbcL and trnL-trnF contained 787 and 930 characters, respectively. For atpB-rbcL, 4 nucleotide positions were variable and 5 were parsimony-informative. For trnL-trnF, 56 were variable and 6 were parsimony-informative. Maximum parsimony produced a MP tree with 72 steps.
Bayesian analysis produced a tree was not significantly different from the MP tree, and the Bayesian tree was shown in Figure 2. The five individuals of Prunus pananensis formed a clade that is sister to P. schneideriana. The clade of P. pananensis+P. schneideriana was well supported with 84% boostrap support, and thus P. pananensis is supported as more closely related to P. schneideriana than to P. discoidea.
The molecular evidence indicated that the individuals of Prunus pananensis formed an independent clade and fit the phylogenetic species concept [20][21][22]. Further, the new species and the sister species are reciprocally monophyletic. The morphological observations showed that Prunus pananensis also fit the diagnosable species concept and the traditional phenetic/morphological species concepts [1]. Trees deciduous, 2.4-8 m tall. Bark brown, with grayish brown or grayish white lenticels. Young shoot densely covered with yellowish brown villose trichomes, becoming brown-pubescent later. Winter buds ovoid, glabrous. Leaf blades obovate-elliptic, elliptic or oblong, chartaceous, 4-10(-12) cm long, 2-4.5(-6) cm wide, caudate, rarely acuminate at apex, rounded or slightly cordate at base, margin acutely serrulate and teeth with a minute disciform apical gland, adaxially greenish yellow to yellowish brown (when dried), almost glabrous, abaxially brown (when dried), and costa and lateral veins densely covered with yellowish brown villose trichomes; lateral veins 8-10 pairs, curved. Petiole 6-12 mm long, densely covered with yellowish brown villose trichomes, with one pair of glands at apex. Stipules narrowly linear, 8-19 mm long, deeply lobed, margin pectinate and teeth with a minute conical apical gland. Inflorescences umbellate, 2 or 3-flowered, rarely solitary, with scales at base; scales leathery, purplish brown, broadly ovate, 3-5 mm long, 2.5-4 mm wide, glabrous on both surfaces; involucral bracts green, apex brown, obovate, 6-8 mm long, 5-6 mm wide, obtuse and serrate-lobed at apex, margin with minute glands, adaxially hirsute, densely at upper part, abaxially glabrous; peduncles 6-10 mm long, concealed in scales or slightly exserted, spreading pilose; bracts green, suborbicular, 3-4 mm in diam., leathery, sparsely pilose, margin with minute disciform glands; pedicels 8-12 mm long, spreading pilose. Flowers opening before or with leaves; hypanthium campanulate, ca. 3 mm long, 2-2.5 mm wide, pilose; sepals ovate-oblong, reflexed, 5-6.5 mm long, 2-3 mm wide, acuminate or acute at apex, outside sparsely pilose at margin, inside glabrous; Nomenclature: -The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants [23,24], and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.

Taxonomic treatment
In addition, new names contained in this work have been submitted to IPNI, from where they will be made available to the Global Names Index. The IPNI LSIDs can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix http://ipni.org/. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS.