Begonia parvibracteata , a new species in Begonia sect. Platycentrum (Begoniaceae) from Guangxi of China, based on morphological and molecular evidence

The previously reported begonias in a limestone forest of Guangxi mainly belong to Begonia sect. Coelocentrum Irmscher. In this article, we described and illustrated a new species in sect. Platycentrum (Klotzsch) A.DC., Begonia parvibracteata X.X.Feng, R.K.Li & Z.X.Liu, which was discovered in a karst forest of south-western Guangxi. The begonia shows high morphological similarity to B. subhowii S.H. Huang and B. psilophylla Irmscher, but differs from the latter two in its narrower oblique-ovate asymmetric leaf blade, 4 (occasionally 6) tepals of pistillate flower and smaller membranous inflorescence bracts. Molecular phylogenetic analysis, based on ITS sequence data, supports the new species as monophyletic and distinct from B. subhowii and B. psilophylla . Considering its narrow distribution and the disturbance of human activities, the conservation status of new taxon is evaluated as “Vulnerable” (VU B1, B2 ab (i, iv, v), D2) according to the IUCN Red List Categories and Criteria.


Introduction
According to the latest report, China's wild begonias have already increased to 239 species (iBegonia 2021). The total number of species could reach 300 in the coming years, meeting the prediction of Tian et al. (2018). Consisting of 122 species, Sect. Platycentrum (Klotzsch) A.DC. represents the largest section of Begonia in China, with 66 species mainly distributed in Yunnan. Eighty-seven species of Begonia have so far been found in Guangxi (iBegonia 2021), most of which (60 species) are distributed in the karst forest and belong to B. sect. Coelocentrum Irmscher.
An unknown Begonia taxon with reproductive organs was collected from Guangxi during our field survey and plant collection in May 2020 and October 2021, respectively. The begonia definitely belongs to B. sect. Platycentrum, but differs markedly from the reported 11 begonias of the same section in Guangxi. After further detailed morphological observation, morphological comparison with similar species and molecular analysis, it is confirmed as a new species in Begonia sect. Platycentrum.

Taxonomic observation
Morphological characters were observed and measured from fresh samples in the field. Morphological comparisons with similar taxa were undertaken by consulting the literature, examining herbarium (IBK and IBSC) specimens and observing living collections cultivated in the nursery of Enshi Dongsheng Plant Development Co. Ltd. The specimens were deposited at the Herbarium of South China Botanical Garden (IBSC), CAS and Chenshan Herbarium (CSH) of Shanghai Chenshan Botanical Garden.

DNA sequencing and molecular analysis
The fresh leaves of the putative new species and the morphologically similar allied species, B. subhowiii and B. psilophylla, were collected in the field and the nursery, respectively. Total DNA was extracted from nine individuals of the three species (B. parvibracteata, B. subhowiii and B. psilophylla) with the CTAB method (Doyle and Doyle 1987). The internal transcribed spacers 1 and 2 (ITS) and the 5.8S gene were amplified using the primers 17SE and 26SE (Forrest and Hollingsworth 2003). PCR amplification and Sanger sequencing were performed according to Fan et al. (2014), with an annealing temperature of 53 °C. The ITS sequences of the three species were uploaded to GenBank (http://www.ncbi.nlm.nih.gov/) with the accession numbers OL892048, OL892049 and OL892050 for B. parvibracteata sp. nov., OL871361, OL871362 and OL871363 for B. subhowii and OL851701, OL851702 and OL851703 for B. psilophylla (Table 1).
We chose 17 species from sect. Platycentrum and 8 species from other sections of Begonia native to mainland China to place the new species in a phylogenetic context (Table 1). Begonia socotrana Hook.f in sect. Peltaugustia (Warb.) Barkley from Socotra (for the coast of Africa) was selected as an out-group in the phylogenetic analysis (Moonlight et al. 2018). Except for the nine individuals of the putative new species and its two allied species, the additional ITS sequences for 23 Begonia species were downloaded from the NR database of NCBI. All these sequences were aligned using BioEdit v.7.2.5 (Hall 1999) and a phylogenetic analysis using Bayesian Inference (BI) was undertaken in MrBayes v.3.1.2 (Ronquist and Huelsenbeck 2003). The GTR+G model was chosen as the optimal model of nucleotide substitution according to the Akaike Information Criterion (AIC; Burnham and Anderson 2002) as implemented in MrModeltest 2.3 (Nylander 2004). The Markov chains were run for 1,000,000 generations and sampled at each 100 generations, with the first 25% discarded as burn-in. Diagnosis. Begonia parvibracteata morphologically resembles B. subhowii and B. psilophylla in rhizome and leaf characters. However, it has narrowly oblique-ovate asymmetric leaf blades, 4 (rarely 6) tepals in pistillate flowers and small (6-8 × 3-5 mm) bracts in inflorescences. These characters differ from the widely ovate leaf blades, 5 (rarely 6) tepals of pistillate flowers, and distinctly large bracts in inflorescences of B. subhowii. B. parvibracteata is dissimilar to B. psilophylla in its 4 (rarely 6) tepals in pistillate flowers and asymmetric, narrowly oblique-ovate leaf blade. Begonia parvibracteata forms a monophyletic group clustered with B. cucurbitifolia in the phylogenic tree, but the latter has nearly symmetric, 3-4 lobed leaf blade, 5-tepaled pistillate flower, definitely differing from the new species.
Distribution and habitat. Currently known from four localities in Longzhou County and Daxin Countym Guangxi, China. It usually grows on rocks or rock cracks in limestone under forest.
Phenology. Flowering September-October, fruiting October-December. Etymology. The specific epithet "parvibracteata" refers to the short small bracts of the new species. The Chinese name is given as "小苞秋海棠" (Begonia with small inflorescence bracts).
Conservation status. There are three populations with approximately 1000 individuals found in Longzhou County. Another one population with approximately 500 individuals is distributed in the Encheng National Nature Reserve, Daxin County. Some plants of this begonia are over-collected and sold in the local medicinal herb market of Longzhou County. According to the IUCN Red List Categories and Criteria (IUCN 2022), B. parvibracteata should be assessed as "Vulnerable (VU B1, B2 ab (i, iv, v), D2)" due to its narrow distribution and the disturbance by human activities.

Molecular analysis
The aligned matrix of the ITS sequence data was 727 bp long. The result of Bayesian Inference analysis is shown in Fig. 4. Begonia sect. Platycentrum appears monophyletic with a high Bayesian posterior probability (bpp = 1) (Fig. 4). The three samples of the putative new species form a monophyletic group clustered with B. cucurbitifolia (bpp = 0.89). Begonia subhowii with the highest morphological similarity to B. parvibracteata formed another subclade.
The nodes with bpp < 0.50 have been collapsed. Sectional placement of taxa is indicated by the following abbreviations: Coe (Coelocentrum), Dip (Diploclinium), Pla (Platycentrum), Rei (Reichenheimia) and Pel (Peltaugustia). The numbers after the species names indicate different populations. The samples of the new species are marked with stars.

Discussion
The new begonia with 2-loculed ovary, axillary placentation and bifid placentae can be clearly assigned to B. sect. Platycentrum (Gu et al. 2007). Within this largest section for Begonia in China, B. parvibracteata shows high morphological resemblance to B. subhowii and B. psilophylla, both of which are distributed in south-eastern Yunnan (Shui and Huang 1999;Shui and Chen 2018), including creeping rhizome, glabrous plants with both basal and cauline leaves at anthesis, usually almost entire leaf margin, palmate venation, fleshy green leaf blade and 4 tepals of staminate flower (Table 2).
In B. subhowii, there are 5 (occasionally 6) tepals in the pistillate flower and the bracts of inflorescence is distinctly long and large (5 × 3 cm) (Fig. 5). These characters differ from 4 (occasionally 6) tepals and triangular to widely ovate (6-8 × 3-5 mm) bracts in B. parvibracteata. In addition, the leaf blade is wider ovate in B. subhowii, compared with the narrower oblique-ovate shape of B. parvibracteata.  In B. psilophylla, the leaf blade is nearly symmetric, widely ovate with symmetric cordate base, in contrast with the narrower ovate leaf blade and oblique-cordate base of the new species. Furthermore, there are 5 (occasionally 6) tepals in the pistillate flower of B. psilophylla, being different from the 4 (occasionally 6) tepals of B. parvibracteata.
According to the phylogenetic tree, based on ITS sequences, the closest relative of B. parvibracteata is B. cucurbitifolia which is also distributed in south-eastern Yunnan. However, B. cucurbitifolia is remarkably distinct from B. parvibracteata in morphology, including nearly symmetric, 3-4 lobed leaf blade and 5-tepaled pistillate flower.
For the flowering time, B. parvibracteata blooms later compared with its three allied species mentioned above.