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Six new species of Begonia from Guangxi, China

Background With currently 1980 described species, the mega-diverse Begonia is now perhaps the 5th largest flowering plant genus, expanding rapidly from ca. 900 species in 1997 to its current size in merely two decades. In continuation of our studies of Asian Begonia, we report six additional new species from Guangxi, the region/province harboring the second richest Begonia flora of China. Results Based on morphological and molecular data, the new species B. aurora belongs to Begonia sect. Platycentrum, while the other five new species (viz. B. larvata, B. longiornithophylla, B. lui, B. scabrifolia, and B. zhuoyuniae) are members of Sect. Coelocentrum. Somatic chromosome numbers of B. longiornithophylla and B. zhuoyuniae at metaphase were counted as 2n = 30, consistent with previously reports for Sect. Coelocentrum. Conclusions With the addition of the six new species, the total number of Begonia species in Guangxi increases from 86 to 92. Detailed description, line drawings, and color plates are provided to aid in identification.


Background
With currently 1980 accepted species (Hughes et al. 2015), the mega-diverse genus Begonia L. is the fastest growing and now perhaps the fifth largest flowering plant genus (Moonlight et al. 2018), doubling from ca. 900 species in 1997 (Frodin 2004) to the current size in merely two decades. One of the major impetus of Begonia's phenomenal growth in the past decades has been the passion and dedication of Dr. Ching-I Peng (Fig. 1) and his collaborations with Begonia researchers and enthusiasts around the world; together the effort has resulted in 81 publications and 98 new Begonia species (Chung 2020). Sadly, Dr. Peng died of illness on 1 May 2018 and his prolific career of Begonia research was ended prematurely (Chung 2020), leaving numerous unfinished works.
To continue his enduring legacy of Begonia research, we here report six new Begonia species that were discovered during Dr. Peng's field trips to Guangxi, China. 2014). Molecular phylogenetic analyses were conducted to further assure their sectional placements.

Morphological observations
Rhizomes of the six new species collected in the field were cultivated in the experimental greenhouse of the Biodiversity Research Center, Academia Sinica, Taipei, Taiwan. Full grown plants with flowers and fruits were used for morphological observation and comparison with morphologically similar species.

Phylogenetic analyses
DNA sequences of three non-coding plastid DNA regions, ndhA intron, ndhF-rpl32 spacer, and rpl32-trnL spacer, were used for phylogenetic analysis according to Moonlight et al. (2018), the most comprehensive phylogenetic study of Begonia up to date. DNA sequences of B. scabrifolia were generated by PCR and the remaining five new species were retrieved from full plastome sequences assembled for our ongoing phylogenomic projects of Begonia using the genome skimming approach (Twyford and Ness 2017). Additionally, DNA sequences of the three regions were also obtained from whole plastom sequence of B. bamaensis that is morphologically similar to B. scabrifolia.
High quality genomic DNA was extracted using the DNeasy Plant Mini kit (Qiagen, Germany). The quantity and quality of DNA were then measured by Qubit TM 3.0 Fluorometer (Thermo Scientific, MA, USA) and by NanoDrop TM 2000 spectrophotometer (Thermo Scientific, MA, USA), respectively. For B. scabrifolia, PCR amplification and DNA sequencing followed Thomas et al. (2011). For the remaining five new species and B. bamaensis, the genomic DNA was sent to whole genome shotgun sequencing (Illumina Hiseq, 250 bp paired-end reads) in the High Throughput Genomics Core at Biodiversity Research Center, Academia Sinica (BRCAS). The sequencing quality of the raw reads were then evaluated by FASTQC v0.11.5 (Andrews 2010). Low quality portions of the reads were trimmed and filtered out by Trimmomatic 0.36 (Bolger et al. 2014). Subsequently, the published plastome sequences of Begonia (Harrison et al. 2016) were used as reference to perform referencebased assembly using the option "Map to Reference" in Geneious Prime (Kearse et al. 2012) to generate a daft genome. Reads were subsequently mapped back to the draft genome to check if there were any problematic assemblies which might be resulted from some minor structural differences between our data and the references. By extracting the correct regions (as contigs) and mapping reads on them to extend these contigs, the problematic parts could be resolved. These extended contigs were mapped back to the draft sequence to correct the assembly, generating a circular genome. The completely assembled plastomes were annotated by GeSeq (Tillich et al. 2017) web tool based on plastomes of Begonia (Harrison et al. 2016). The start and stop codons of each gene were manually checked and adjusted under Geneious Prime. The tRNA genes were further checked by referring to the secondary structures drawn by tRNAscan-SE web server (Chan et al. 2019).
In addition to the six new species and B. bamaensis, DNA sequences of 80 additional Asian Begonia species were downloaded from NCBI (Additional file 1), including one species of Sect. Alicida ( §ALI), six species of Sect. The three plastid sequences were concatenated using amas-0.93 (Borowiec 2016). Sequences were aligned using MAFFT (Katoh and Standley 2013). The final alignment is available as the Additional file 2. The maximum likelihood analyses with 1000 bootstrap resampling were conducted using RAxML-HPC (Stamatakis et al. 2008), with a gamma model of rate heterogeneity and the substitution model GTR + G+I.

Distribution and ecology
Begonia aurora is known only from the type locality where less than 50 plants were seen. Plants grow on a slope of a shaded gully in a mixed forest of broadleaved woods and bamboo plantation.

Phenology
Flowering from March to April, fruiting from April to June.

Etymology
The species epithet refers to the lighter green patch around the midrib of the leaves resembling aurora, the polar lights.

Notes
Our phylogenetic analyses placed Begonia aurora in Sect.

Distribution and ecology
Begonia larvata is only known from the Zuozhou Town, Jiangzhou District, Chongzuo City, Guangxi, growing on semi-shaded limestone cliff surfaces or steep slopes, at 125-165 m in elevation.

Etymology
The species epithet 'larvata' (masked), refers to the leaf variegation that resembles the facial mark of masked palm civet (Paguma larvata).

Chromosome cytology
Somatic chromosomes at metaphase of Begonia longiornithophylla were counted as 2n = 30 (Fig. 9a), identical to the majority of species of Sect. Coelocentrum (Chung et al. 2014;Han et al. 2018). The length of chromosomes varied from ca. 1.1 to 1.6 µm long. Although several longer chromosomes were metacentric and/or submetacentric, the centromere positions of most chromosomes could not be determined. Satellites were not observed.

Distribution and ecology
Southwestern Guangxi, China. On forest floor, creeping on limestone rocks or cliffs in broadleaf forest.

Phenology
Flowering from February to May. Fruiting from May to July.

Etymology
The species epithet refers to its resemblance to Begonia ornithophylla Irmsch., distinct from the latter by its elongated rhizomes.

Notes
Begonia longiornithophylla somewhat resembles B. ornithophylla in the leaves and B. auritistipula in its elongated rhizomes; the new species can be easily distinguished from the two species by several characters such as densely villous and elongated rhizomes, boat-shaped stipules, glandular-pilose peduncles, red-pilose on tepals and ovaries. A detailed comparison of the three species is provided in Table 2.  Liu, Hai-Shan Gao, Kuo-Fang Chung, Ming-Chao Yu, and Lu-Shi Nian (holotype: IBK;isotype: HAST-144968).

Distribution and ecology
Known only from the type locality in Guangxi, China. On limestone hill.

Phenology
Flowering from March to May; fruiting in May.

Distribution and ecology
Begonia scabrifolia has long been cultivated in Guilin Botanical Garden, Guangxi. Its precise origin is not known.

Etymology
The species epithet refers to the rough and scabrous leaf surface of the adaxial side.

Chromosome cytology
Somatic chromosomes of Begonia zhuoyuniae at metaphase were counted as 2n = 30 (Fig. 9b), identical to the number reported by Han et al. (2018) as well as majority of species of Sect. Coelocentrum (Chung et al. 2014). The length of chromosomes varied from ca. 0.8 to 1.3 µm. The centromere positions of several small chromosomes could not be determined. However, most chromosomes were metacentric. Satellites were not observed.

Distribution and ecology
Known only from two limestone caves in Donglan County and Bama Yao Autonomous County, northwestern Guangxi.

Phenology
Flowering from February to May; fruiting from April to July.