Begonia jinyunensis (Begoniaceae, section Platycentrum), a new palmately compound leaved species from Chongqing, China

Continental China is the center of Begonia species diversity in Asia and contains more than 60 species out of about 110 named species of section Platycentrum. Mt. Jinyun, located in Chongqing City at the upper reaches of the Yangtze River, harbors a subtropical broadleaved forest with high species diversity. During a botanical survey in Mt. Jinyun, an unknown Begonia species of sect. Platycentrum with palmately compound leaves was collected and studied based on detailed morphological observations and cytological and molecular phylogenetic analyses. The unknown Begonia bears a superficial resemblance to B. hemsleyana in having palmately compound leaves, a feature unseen in other species of sect. Platycentrum in China. It is however sharply distinct from the latter in the acaulous habit with aerial stems seen only at anthesis and long rhizomes (vs. erect stems to 70 cm or taller with short rhizomes), 4–6 pinnatilobed leaflets with indistinct, decurrent petiolules (vs. 7–10 serrate leaflets with distinct petiolules), and white (vs. pink) tepals. Molecular phylogenetic analyses based on nuclear ribosomal DNA and chloroplast DNA sequences indicated that this species was allied to Platycentrum species occurring in Southwest and South-central China and Vietnam, including B. hemsleyana, and clearly separable from these species. Somatic chromosome number of 2n = 22 was reported for this unknown species. The diploid chromosome number is agreeable with those published for Begonia sect. Platycentrum. The unknown Begonia is confirmed to be a new species of sect. Platycentrum and hereto described as Begonia jinyunensis C.-I Peng, B. Ding & Q. Wang.


Background
The mega-diverse genus Begonia L. (Begoniaceae) comprises ca. 1,500-1,600 species widely distributed in the tropical and subtropical regions of the world except Australia (Kiew 2005, Tebbitt 2005, Chung et al. 2014. Section Platycentrum (Klotzsch) A. DC. comprises about 110 species of herbs and subshrubs that are widely distributed in Asia, ranging from India to the Himalayas, Indo-China, China, Taiwan, and Malesia (Doorenbos et al. 1998, Shui et al. 2002. Continental China is the center of Begonia species diversity in Asia and contains more than 60 Platycentrum species predominantly in South of Yangtze River (Shui et al. 2002, Gu 2007, Wei et al. 2007. They are either terrestrial on thick humus, epiphytic on trees, or epipetric on rock faces or crevices at 100-2,200 m altitude (Shui et al. 2002). Many Begonia species are local-or site-endemics and even after the publication of Flora of China ) many new Begonia species have been discovered (Liu et al. 2007, Shui 2007, Wei et al. 2007, Ku et al. 2008, Li et al. 2008, Peng et al. 2008a, 2008b, 2010, 2013, 2014, suggesting that the species-rich genus has not been fully explored in China. Mt. Jinyun (29°49' N, 106°20' E; Figure 1) is located at the upper reaches of the Yangtze River and has the highest peak (950 m) in Chongqing Municipality, Southwest China. The climate is subtropical monsoon, with annual mean temperature of 18.3°C and annual rainfall of 1,105 mm, and the vegetation is mixed evergreen broadleaved and conifer forest, with patches of bamboos (Qing et al. 2004). The area, although only 20 km away from the center of the megacity, harbors a typical forest landscape of the Yangtze River basin and high species diversity, and is protected as Jinyun Mountain National Nature Reserve with the core area of 1,235 hectares (Qing et al. 2004). In a botanical survey of the nature reserve in July 2013, Bo Ding, Qian Wang, and Hai-Jun Wen collected an unknown Begonia of sect. Platycentrum with palmately compound leaves and sent it to Ching-I Peng for cultivation at the experimental greenhouse in Academia Sinica for careful examination. In Begonia sect. Platycentrum, only one species, B. hemsleyana Hook. f. occurring in Southern China (Southwest Guangxi and Southeast Yunnan) and northern Vietnam , has palmately compound leaves. Based on detailed morphological study, both of herbarium materials and living collection, and cytological and molecular phylogenetic analyses, we confirmed that the unknown Begonia is a new species, which is hereby described as Begonia jinyunensis C.-I Peng, B. Ding & Q. Wang.

Morphological study
Living collections of Begonia jinyunensis (Bo Ding 20130701, Ching-I Peng 24108, 24109, 24110) were used for morphological observations. In addition, herbarium specimens representative of B. jinyunensis were carefully studied in IBSC.

Molecular phylogenetic analyses
Molecular phylogenetic analyses of B. jinyunensis incorporated B. hemsleyana and 21 species (Appendix 1); these 21 species fell in the same clade with B. hemsleyana in a previous phylogenetic study on Asian Begonia based on concatenated sequences of the internal transcribed spacer (ITS) region (including ITS1 and ITS2 and the 5.8S rRNA gene) of 18S-26S nuclear ribosomal DNA (nrDNA) and an intron of rpl16 gene (rpl16) of chloroplast DNA (cpDNA) (Chung et al. 2014). These species were mostly sect. Platycentrum (14 species including B. hemsleyana) but also included five species of sect. Diploclinium (Lind.) A. DC., two of sect. Sphenanthera (Hassk.) Warb., and B. longicarpa K. Y. Guan & D. K. Tian, currently not assigned to any section (Chung et al., 2014). Note that Asian sections of Begonia are largely non-monophyletic (Thomas et al. 2011, Chung et al. 2014 I Peng 17590, 20933, 22778) and one from Vietnam (Ching-I Peng 20204), were studied. For the other species one sample each was used. Samples sequenced in this study are indicated with asterisks (Appendix 1) and sequences of the other samples were cited from Chung et al. (2014). Methods of DNA extraction, PCR, and DNA sequencing followed Nakamura et al. (2012). DNA sequences were aligned using ClustalX ver. 1.8 (Thompson et al. 1997) and then manually adjusted. Phylogenetic analyses were based on a Bayesian approach using MrBayes ver. 3.1.2 (Ronquist & Huelsenbeck 2003) and a maximum parsimony (MP) criterion using PAUP* ver. 4.0b10 (Swofford 2002).
In the Bayesian phylogenetic analysis, the best substitution models for the ITS and rpl16 data were selected as SYM + I and GTR + I, respectively, using KAKUSAN4 (Tanabe 2011) based on Bayesian information criterion (BIC). Two separate runs of Metropolis coupled Markov chain Monte Carlo analyses were performed, each with a random starting tree and four chains (one cold and three heated). The chain length was ten million generations, and the chain was sampled every one thousandth generation from the cold chain. The mixing and convergence of the chains of the two runs was assessed using Tracer ver. 1.5.0 (Drummond & Rambaut 2007). The first 10% of the total 10,000 sample trees were discarded as burn-in. After the burn-in, the effective sample sizes of all parameters were > 200, indicating that the analyses sampled the posterior distributions of each parameter satisfactorily, and the values of Average Standard Deviation of Split Frequency (ASDSF) were below 0.005. The 50% majority rule consensus tree of all the post-burn-in trees with Bayesian posterior probabilities (PP) was visualized with FigTree ver. 1.3.1 (Drummond & Rambaut 2007).
In the MP phylogenetic analysis, indels were treated as missing data. The characters were treated as unordered, and the character transformations were equally weighted. The branch collapse option was set to collapse at a minimum length of zero. A heuristic parsimony search was performed with 1,000 replicates of random additions of sequences with ACCTRAN character optimization, tree bisection-reconnection (TBR) branch swapping, and MULTREES and STEEPEST DESCENT options on. Statistical support for each clade was assessed by bootstrap analysis (Felsenstein 1985). Ten thousand replicates of heuristic searches, with the TBR branch swapping switched on and MULTREES options off, were performed to calculate bootstrap percentages (BP).

Chromosome cytology
Somatic chromosome morphology was studied for one plant of B. jinyunensis (Ching-I Peng 24110, HAST) using root tips. The methods of pretreatment, fixation, and staining for chromosome observations followed our previous paper . Classification of the chromosome complements based on centromere position at mitotic metaphase followed Levan et al. (1964).

Molecular phylogeny based on nrDNA and cpDNA
The aligned length of the ITS and rpl16 sequences was 695 bp and 1,002 bp, respectively. In the concatenated data set, 323 nucleotide substitutions were found in 277 variable sites and 120 sites were parsimony informative among them. The Bayesian 50% majority rule consensus tree with mean branch length and PP is depicted (Figure 2). The MP analysis yielded 44 equally parsimonious trees of 641 steps with a consistency index (CI) = 0.682, a retention index (RI) = 0.614, and a rescaled consistency index (RC) = 0.418. Clades supported with BP ≥ 50% in the MP strict consensus tree (not shown) were all recognized in the Bayesian tree. BP was plotted on the Bayesian tree. Only clades supported by PP ≥ 0.95 and/or BP ≥ 70% were considered adequately supported. Begonia jinyunensis resided in a clade with B. hemsleyana, B. augustinei, B. dryadis, B. edulis, and B. pedatifida (PP = 0.95/BP < 50%). These are species of sect. Platycentrum in Southwest and South-central China and Vietnam ). Although phylogenetic relationship among the species within this clade was not fully resolved except for the clade of B. augustinei and B. dryadis (0.97/< 50%), the six species were clearly separated from each other, and B. jinyunensis (1.0/100%) and B. hemsleyana (1.0/99%) were reciprocally monophyletic.

Chromosomal features
Somatic chromosomes at metaphase of B. jinyunensis were determined to be 2n = 22 (Figure 3). The 22 chromosomes gradually varied in length from ca. 1.0 to 1.8 μm.

Diagnosis
Begonia jinyunensis bears a superficial resemblance to B. hemsleyana in having palmately compound leaves, a feature unseen in other species of sect. Platycentrum in China. It is however sharply distinct from the latter in the acaulous habit with short aerial stems seen only at anthesis and long rhizomes (vs. erect stems to 70 cm or taller with short rhizomes), 4-6 pinnatilobed leaflets with indistinct, decurrent petiolules (vs. 7-10 serrate leaflets with distinct petiolules), and white (vs. pink) tepals.

Distribution, habitat and ecology
Begonia jinyunensis is currently known only from Jinyun Mountain National Nature Reserve. The species usually grows on limestone faces of shaded moist environments such as valleys and broadleaved forests. Flowering in July; fruiting in August.

Etymology
The specific epithet is derived from the type locality, Mt. Jinyun.

IUCN Red list category
Vulnerable (VU D2). Begonia jinyunensis is known only from a narrow area of Mt. Jinyun, Chongqing Municipality. Although the area is under protection as a national nature reserve, habitat disturbance brought about by human activities such as tourism and maintenance/ building of roads/walking trails may have a negative impact on the species.

Conclusions
Detailed morphological observations and molecular phylogenetic analyses based on nuclear ribosomal DNA and chloroplast DNA sequences supported the recognition of the new species Begonia jinyunensis. Somatic chromosome number of 2n = 22 was reported for this new species. Begonia jinyunensis is currently known only from the type locality, Mt. Jinyun, Chongqing Municipality, Southwest China.