Phylogenetic analyses of Begonia sect. Coelocentrum and allied limestone species of China shed light on the evolution of Sino-Vietnamese karst flora

Taxon sampling

Our sampling strategy aimed to increase the number of species of limestone Begonia of continental China not represented in previous molecular phylogenetic analyses, while maximizing representatives of all major clades in Asia (Thomas et al.2011a). In total, 94 Asian species (44 species of mainland China) were sampled, including one species of sect. Alicida C.B. Clarke (ALI), eight species of sect. Baryandra A. DC. (BAR), 19 species of sect. Coelocentrum (COL), 12 species of sect. Diploclinium (DIP), one species of sect. Haagea (Klotzsch) A. DC. (HAA), four species of sect. Leprosae (T.C. Ku) Y.M. Shui (LEP), one species of sect. Parvibegonia A. DC. (PAR), 25 species of sect. Petermannia (PET), 14 species of sect. Platycentrum (PLA), three species of sect. Reichenheimia (REI), one species of sect. Ridleyella Irmsch. (RID), three species of sect. Sphenanthera (SPH), one species of sect. Symbegonia (SYM), and B. boisiana Gagnep., which is assigned to sect. Petermannia by Kiew (2007) but placed as unassignable (UA) to existing section in Doorenbos et al. (1998) and Hughes and Pullan (2007). Two South African species were chosen as outgroups based on previous phylogenetic analyses (Goodall-Copestake et al.20092010; Thomas et al.2011a). Of the 96 species sampled, 49 species (51%) have never been investigated using molecular data. The majority of the sampled species were field-collected plants maintained in the experimental greenhouse of the Academic Sinica, Taipei, Taiwan. Voucher information and GenBank accession numbers are detailed in Additional file1.

Molecular data

DNA of Begonia species is difficult to extract, especially for those collected from limestone substrates. Additionally, silica-gel dried leaves of Begonia tend to yield poor quality DNA. To circumvent these issues, total genomic DNA was extracted from fresh leaves of living collections using a modified CTAB protocol optimized for Begonia (Kopperud and Einset1995).

Two DNA sequence regions were used: the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) region and the chloroplast DNA rpL16 intron. Previous analyses based on ITS (e.g., Tebbitt et al.2006) suggested its usefulness in resolving interspecific relationships while chloroplast sequences were more suitable for reconstructing the backbone structure of the Asian Begonia phylogeny (Thomas et al.2011a). Although the rpL16 intron has thus far never been used in reconstructing the Begonia phylogeny, this region amplified and sequenced easily and shows adequate phylogenetic resolution in Asian Begonia. For each polymerase chain reaction (PCR), amplification was performed in a total volume of 25 μl, including 12.5 μl of Taq DNA Polymerase Master Mix Red (Ampliqon, Copenhagen, Denmark), 1 μl of each forward and reverse primer (10 μM), 2 μl of template DNA, and 8.5 μl of ddH₂O. For ITS, the primers 5P and 26S1Rev primers (Clement et al.2004) were used for both PCR amplification and sequencing. For rpL16 intron, three primers (rpL16-F: GCT ATG CTT AGT GTG TGA CTC G; rpL16-R: CGT CCY GCT TCT ATT TGT CTA G; Beg_rpL16: GTT TCA CAT TAT CTG GAT CG) were designed to optimize PCR amplification (rpL16-F and rpL16-R) and sequencing (Beg_rpL16 and rpL16-R) in Begonia. PCR reactions were carried out by a denaturation-step in 94°C for 5 min, 30 thermo-cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 90 s (60 s for ITS), and a final extension in 72°C for 5 min. PCR products were purified using QIAquick PCR purification Kit (Qiagen, Valencia, California, U.S.A.) and then sequenced using an ABI PRISM dye terminator cycle sequencer, model 3700 (Applied Biosystems, Foster City, California, U.S.A.).

Phylogenetic analyses

DNA sequences were aligned using the program MUSCLE implemented in the software MEGA5 (Tamura et al.2011) with subsequent manual adjustments. The aligned matrix was analyzed using maximum parsimony (MP) and maximum likelihood (ML) optimality criteria and Bayesian Inference (BI). MP analyses were performed using PAUP* v.4.0b10 (Swofford2002). All characters were unordered and equally weighted and gaps were treated as missing. Heuristic searches were conducted with 1000 replicates of random addition, 10 trees held at each step during stepwise addition, tree-bisection-reconnection (TBR) branch-swapping, and deepest descent option in effect. Brach supports (parsimony bootstrap: PB) were assessed by 1000 bootstrap replicates with 10 random taxon additions and heuristic search options. To assess the congruence among the two data sets, the incongruence length difference test (ILD test for partition-homogeneity test in PAUP*) was performed. One thousand homogeneity replicates of heuristic searches were conducted with 100 random sequence additions and MulTrees not in effect. Ten ML analyses with 5000 bootstrap (likelihood bootstrap: LB) resamplings were conducted using RAxML-HPC2 v7.2.7-3 (Stamatakis et al.2008) via the CIPRES Portals (http://www.phylo.org/index.php/portal/). The matrix was partitioned (i.e., ITS vs. rpL16 intron) with a gamma model of rate heterogeneity and proportion of invariable sites estimated by the program. The program MrBayes v3.1.2 (Ronquist and Huelsenbeck2003) was employed for BI analyses. The aligned matrix was partitioned into ITS and rpL16 data sets. Prior to the analysis, DNA substitution models for each partition were selected by MrModeltest (Nylander2004) based on the Akaike information Criterion (AIC). BI analyses were conducted using mixed models specified to each data partition. Four runs of Metropolis-coupled Markov chain Monte Carlo (MCMCMC) analyses were performed, with a random starting tree and four chains for each run (one cold and three heated). The MCMCMC length was ten million generations and the chain was sampled every 1000th generation from the cold chain. To assure that the cold chain swapped successfully with the heated chains and the Metropolis coupling functioned well, the resulting log files were by inspecting the trace plots of parameters using the program Tracer v1.5 (Drummond and Rambaut2007). The function ‘Compare and Cumulative’ of the program AWTY (Nylander et al.2008) was employed to check topological convergence and convergence of posterior probabilities (PP) of splits from different runs. Bayesian clade posterior probabilities and average branch lengths were calculated based on the sampled trees after the first 25% of the sampled trees were discarded as burn-in.

Molecular dating

The software package BEAST v1.6.1 (Drummond and Rambaut2007) was employed to perform divergence-time estimates. The aligned matrix was formatted for BEAST using BEAUti with the data partitioned to allow specification of DNA substitution models for each partition (see BI analysis above). Because suitable fossils in Begoniaceae are not available, the phylogenetic tree was calibrated using two normally distributed priors based on divergence time estimated by Thomas et al. (2012) for Asian Begonia: the mean of estimated divergence ages of the crown node of Asian Begonia (Clade 10 in Figure three of Thomas et al.2012; the node marked with a rhombus sign in Figure 1) at 16.1 (95% Highest Posterior Density range [HPD] 9.1–22.5) mya (million years ago) and Clade 49 in Figure three of Thomas et al.2012 (clade D, marked with a star sign in Figure 1) at 13.0 (7.7–18.9 HPD) mya. Clade D in Figure three of Thomas et al. (2012) includes sect. Coelocentrum and Petermannia, and species of sect. Diploclinium (Clade BAR) centered in the Philippines recently classified under the recircumscribed sect. Baryandara (Rubite et al.2013). For these two clades, the calibration priors were modeled with the mean of 16.1 mya and the standard deviation of 3.265 (95% probability interval: 9.7–22.5 mya) for the Asian Begonia Clade and the mean of 13.0 mya and the standard deviation of 2.7 (95% probability interval: 7.7–18.3 mya) for Clade 49 to account for HPDs of age estimates of the original analysis. The tree prior was set to the Yule process and a single uncorrelated-rates relaxed molecular clock model assuming a lognormal distribution of rates (UCLD) was applied to all partitions. Two independent runs of BEAST analyses were conducted, each with 50 million generations and sampling every 1,000th generation.

Taxonomic implications of the phylogenetic analyses

Based on a very different set of taxonomic sampling, our analyses corroborate results of previous analyses (Tebbitt et al.2006; Thomas et al.2011a) in confirming the non-monophyly of most major Asian sections, including sect. Diploclinium, Leprosae, Petermannia, Platycentrum, Reichenheimia, and Sphenanthera (Figure 1). Additionally, our expanded sampling of Chinese Begonia species further demonstrates the paraphyly of sect. Coelocentrum, dominating in the strongly supported Clade SVLB otherwise also composed of two species of sect. Diploclinium (B. cavaleriei and B. pulvinifera), two species of sect. Leprosae (i.e., B. cylindrica and B. leprosa), and one species of sect. Petermannia (B. sinofloribunda). Despite being one of the biggest (47 species) and morphologically most diverse sections in China, only three rhizomatous species of sect. Diploclinium (B. cavalerieri, B. pulvinifera, and B. wangii) are known from the Sino-Vietnamese limestone karsts (Gu et al.2007; Peng et al.2006), none sampled in previous phylogenetic analyses. Our study thus further strengthens the polyphyly of sect. Diploclinium and the homoplasious nature of its diagnostic characters, i.e., bilamellate, 3-locular axillary placentation, in Asian Begonia (Thomas et al.2011a).

Sect. Leprosae, raised for three species (B. cylindrica, B. leprosa and B. longicarpa) bearing cylindric and berry-like fruits in southern China (Shui et al.2002), has been questioned to be artificial when B. bataiensis Kiew, a tuberous species bearing the characteristic fruit type from limestone karsts of southwestern corner of Vietnam described (Tam et al.2005). Subsequently molecular study sampling two species (B. leprosa and B. longicarpa) showed that sect. Leprosae is polyphyletic with its type species B. leprosa (limestone species) placed sister to sect. Coelocentrum and B. longicarpa (not limestone species) nested within clade dominated by sect. Platycentrum and Sphenanthera, suggesting that the cylindric and berry-like fruits evolved independently (Tebbitt et al.2006). With all four recognized species sampled, our analyses attested further the polyphyly of sect. Leprosae, with two limestone species (B. cylindrica and B. leprosa) in the Sino-Vietnamese karsts nested within Clade SVLB, B. longicarpa in Clade PLA-SPH, and B. bataienssi sister to B. kingiana (Figure 1).

Known only from hills of limestone karsts of the southern Guangxi adjacent to the Vietnamese border (Gu et al.2007), B. sinofloribunda, a rare species uniquely characterized by branched aerial, subwoody stems and pendulous inflorescences with small flowers and ovaries showing axillary placentas, has been variously classified in sect. Platycentrum (Gu2007; Ku1999), Diploclinium (Shui et al.2002 and Petermannia (Shui and Chen2004). Our analysis nevertheless indicates strongly that B. sinofloribunda is derived from within Clade SVLB (Figure 1), greatly increase the morphological diversity of Clade SVLB.

Based on our study, although species of Clade SVLB vary greatly in their ovary anatomy and fruit type that are crucial for sectional classification in Begonia, they are all rhizomatous distributed exclusively in cave-like microhabitats of the limestone karsts of the Sino-Vietnamese bordering areas. Additionally all available data, including an unpublished chromosome count of B. sinofloribunda (Peng, unpublished data), indicate that members of Clade SVLB are uniformly characterized by the chromosomal number 2n = 30 Gu et al. (2007; Peng et al.2010; Thomas et al.2011a) or its triploid number 2n = 45 (Peng et al.2013). Given the obvious cohesiveness in their perennation organs as well as ecology, biogeography and chromosome cytology, we thus propose to expand the definition of sect. Coelocentrum (see Taxonomic Treatment) to include B. sinofloribunda, three species of sect. Diploclinium (B. cavaleriei, B. pulvinifera, and B. wangii; see Taxonomic Treatment) and two species of sect. Leprosae (B. cylindrica and B. leprosa).

Phylogenetics and molecular dating of Clade SVLB

Except for species of Clade SVLB, the only Begonia species found on the Sino-Vietnamese limestone karsts are the tuberous B. chingii Irmsch. and B. grandis (Gu et al.2007) of the DIP I grade (Figure 1) and B. boisiana that possesses upright stems (Tebbitt2005). The phylogenetic relationships therefore indicate that, in the Sino-Vietnamese limestone karsts, Begonia had evolved to adapt to the harsh limestone habitats four times, twice evolved with the tuberous and deciduous habit (or once and secondary invading the none-limestone habitat by B. labordei, Figure 1), once by the species with upright stem (B. boisiana), and once by the rhizomatous ancestor of Clade SVLB (Figure 1). Interestingly, although tubers and a deciduous habit appear to be suitable for surviving in the desiccation-prone limestone habitats such as most karst Begonia in Madagascar (Grubb2003; Keraudren-Aymonin1983), our data strongly suggest that it is the rhizomatous Clade SVLB that succeeded and proliferated in the Sino-Vietnamese limestone karst terrains.

Within the strongly supported Clade SVLB, phylogenetic resolution is poor (Figure 1), possibly indicating a rapid species radiation (Hughes and Eastwood2006; Kozak et al.2006). Based on the divergence times calculated by BEAST, the crown age of Clade SVLB (Clade 35 in Figure 2; Table 2) was estimated to be ca. 8.06 (HPD 5.09–11.21) mya during the middle to late Miocene, with the stem age extending back to ca. 11 mya (Figure 2). Although these age estimates are likely problematic because of the employment of secondary calibration, even within the lowest end (ca. 5 mya), the crown age of the Sino-Vietnamese limestone Begonia is much older than those estimated in recent phylogenetic literature such as species radiations triggered by the Pleistocene uplift of the South American Andes (e.g., Hughes and Eastwood2006; Hoorn et al.2010).

Interestingly, the estimated crown age of Clade SVLB at 8.06 (HPD 5.09–11.21) mya (Table 2; Figure 2) appears to coincide well with the most extensive stage of karstifications in Guangxi during the Miocene (Liu19972003) and the onset and intensification of the East Asian monsoon at ca. 7.2 (An2000) to 11 mya (Zheng et al.2004). In Asia, the climate has been affected strongly by the altitude of the Himalayas and the Tibetan Plateau. By the late Miocene, the coeval uplift of Himalaya-Tibetan region had attained significant heights that greatly altered Asian atmospheric circulation, resulting in the onset of the warm and humid East Asian summer monsoon (An et al.2001). Because high temperature and precipitation brought by the summer monsoon are favorable conditions for the development of karsts (Zhang1989), the late Miocene onset of the East Asian monsoon would have greatly facilitated the development of limestone karsts across the Sino-Vietnamese limestone regions (Liu19972003), resulting in the remarkable landscapes and providing ample habitats for the spreading and diversification of limestone karst plants such as Begonia. Recently the uplift of Himalaya-Tibetan region and intensification of East Asian monsoon have also been suggested to be responsible for triggering the rapid diversification of both seasonally and wet adapted Begonia in continental Asia (Rajbhandary et al.2011) and the fern genus Lepisorus in China and Japan (Wang et al.2012).

Diversity pattern of Clade SVLB

Species of the Clade SVLB, comprising ca. 60 species (see Taxonomic Treatment) mainly distributed in Guangxi, inhabit the ‘twilight zone’ (Poulson and White1969) of limestone caves and cave-like microhabitats such as shaded fissures and crevices abundantly occurring throughout the Sino-Vietnamese limestone karst region (Sweeting1978; Schindler1982; Xu1995; Zhu2007). Such cave-like microhabitats, characterized by constant temperature, high humidity, and indirect and low light (Liang et al.2010), function as shelters on the limestone bedrock characterized by highly alkaline conditions, thin soil layers, and severe desiccation due to high porosity (Clements et al.2006). In addition to Begonia, these cave-like microhabitats are also inhabited by several species-rich, calciphilous herbaceous plant groups such as Aspidistra Ker Gawl. (e.g., Liu et al.2011), a number of genera of gesnerids (e.g., Weber et al.2011; Xu et al.2012a2012b), Elatostema L. (e.g., Wei et al.2011), Impatiens (e.g., Yu et al.2009), and the fern genus Polystichum (e.g., Zhang and He2011), etc. Many of these limestone plants are often highly ornamental and have long been commercially exploited (Vermeulen and Whitten1999). Biogeographically most of these calciphilous cave plant species have a very limited distribution ranges and site-endemics are very common (Liu et al.2007; Peng et al.2008a2012; Xu et al.2012a; Zhang and He2011). In individual caves or crevices, species diversity is often low; however, across the landscapes, plant composition varies greatly from one cave to another, resulting in low α diversity and yet extremely high β diversity across the landscape (Hughes and Hollingsworth2008).

Phylogenetic niche conservatism of Clade SVLB

The monophyly of the rhizomatous Clade SVLB suggests a single evolutionary origin of the adaptation to the cave-like limestone microhabitats, and shows that ecology is good indicator of taxonomic relationships than traditional morphological characters used to defined sections. Inspecting the chronogram (Figure 2) reveals a 3 MY-time lag between the estimated stem and crown age of Clade SVLB, which might signify a long period needed for their ancestor to gradually adapt to the calcium-rich and cave-like microhabitats on the limestone karsts before the commencement of species radiation. Its monophyly and prolonged existence since the late Miocene also suggest the strong tendency in the evolution of this clade to retain the ancestral ecological niche, presenting an apparent case of strong ‘phylogenetic niche conservatism’ (Kozak et al.2006; Wiens2004).

Cave-like microhabitat as paleorefugia

Limestone caves and crevices/fissures are formed through the development of underground hydrological systems with maximum discharge and dissolutional aggressiveness (Palmer1991). The development and ‘collapse’ of caves are essential processes shaping the karst topography (Ford and Williams2007; Palmer1991). In more recent histories, anthropogenic factors have also greatly accelerated the destruction of caves and cave-like microhabitats in the karsts (Clements et al.2006). Together with the observations of the unique ecology of limestone Begonia ,limestone caves and crevices currently inhabited by rich plant communities can be viewed as “biological refugia, habitats that support populations not able to live elsewhere in a landscape (Nekola1999”. More precisely, these cave-like microhabitats in the Sino-Vietnamese limestone karsts qualify as “paleorefugia, now-fragmented relicts of a formerly widespread matrix community (Nekola1999)”. On the contrary, “neorefugia represent habitats that are younger than the surrounding biological matrix (Nekola1999)”.

Clade SVLB as an example of a non-adaptive radiation on limestone karsts

The onset of Asian summer monsoon in the late Miocene (An et al.2001) provided ideal conditions for the karst development (Zhang1989) across the limestone terrains of the Sino-Vietnamese bordering region (Liu1997), resulting in numerous crevices, fissures, and caves and the splendid fenglins and fengcongs across the landscapes. Although limestone karsts are an unsuitable growth substrate for most plants, over time, some plant groups (e.g., Aspidistra, Begonia, Impatiens, various gesnerid genera, Elatostema, etc.) adapted to and flourishing in the cave-like microhabitats. However, cave-like microhabitats are fragile and collapsed constantly due to the processes of karstification (Palmer1991) associated with the persistence of East Asian monsoon (An et al.2001; Liu1997), resulting in fragmentation and isolation of these microhabitats. Moreover, because of the strong niche conservatism (Wiens2004) to the cave-like microhabitats, populations of the calciphilous plants had been forced to track the paleorefugia (Wiens2004; Kozak et al.2006; Comes et al.2008). Moreover, because Begonia species are poor dispersers (Hughes and Hollingsworth2008), gene flow among populations would be retarded, greatly augmenting effects of random genetic drift (Comes et al.2008; Hughes and Hollingsworth2008). As geological weathering processes have occurred continuously throughout the Sino-Vietnamese limestone karsts, population diversification likely would have resulted in steady accumulation of species (Kozak et al.2006; Comes et al.2008; Hughes and Hollingsworth2008). However, because the major driving force of this evolutionary process is geographic isolation, the diversity pattern is better characterized as ‘non-adaptive radiation’, an underappreciated speciation mode crucial for rapid species accumulations in organisms of low vagility and strong niche conservatism (Gittenberger1991; Kozak et al.2006; Comes et al.2008; Rundell and Price2009). Morphologically, species of Clade SVLB all have very similar flowers, distinguishing from one another mainly by vegetative characters such as leaf morphology in shape, pubescence, texture, and variegation (Gu et al.2007). Recent studies, however, show that different variegations in Begonia leaves have no apparent adaptive differences (Sheue et al.2012; Zhang et al.2009), suggesting that the species accumulation in Begonia was mainly a non-adaptive process (Gittenberger1991; Kozak et al.2006; Comes et al.2008; Rundell and Price2009), though further studies are definitely needed to test the non-adaptive nature of leaf morphology in Clade SVLB.

Taxonomic Treatment

Synopsis of Begonia sect. Coelocentrum Irmsch.

Begonia sect. Coelocentrum Irmsch., Mitt. Inst. Allg. Bot. Hamburg 10: 533. 1939; Doorenbos et al., The section of Begonia 84. 1998; Ku, Fl. Reipubl. Popularis Sin. 52(1): 127, 129. 1999; Shui et al., Bot. Bull. Acad. Sin. 43: 314. 2002; Gu, Fl. China 13: 205. 2007. LECTOTYPE SPECIES: B. porteri H. Lév. & Vaniot (designated by Barkley and Baranov1972).—Begonia sect. Leprosae (T.C. Ku) Y.M. Shui, Bot. Bull. Acad. Sin. 43: 321. 2002, syn. nov.

Herbs monoecious, terrestrial, perennial, rhizomatous without erect stems and tubers, acaulescent, rhizome herbaceous or slightly woody. Leaves alternate, variable, mostly broadly ovate, oblique at base, rarely palmate, palmately compound, or occasionally peltate, glabrous to tomentose, often variegated. Inflorescence axillary, cymose (dichasial or dichasial at base and monochasial at apex), rarely thyrsoid, staminate flowers basal and carpellate flowers distal, protandrous. Perianth segments free, usually white or pink, rarely greenish or yellowish. Staminate flower with 4, or rarely 2 tepals. Carpellate flowers usually with 3 tepals, rarely 2 or 5 (–6); ovary 3-winged, or rarely unwinged, wings equal to unequal in fruit, mostly 1-locular, parietal placentation, rarely 3-locular with axile placentation. Fruit a 3-winged capsule, nodding, or rarely berry-like, cylindric.

Cytology. 2n = 30 or rarely 45 (Peng et al.200620072008b201020122013;Gu et al. 2007; Liu et al.2007).

Distribution and Habitat. Currently 61 species, two varieties, and one natural hybrid distributed in the caves, fissures and forest floors of limestone karsts in southern China (Guangxi, southern Guizhou, and southeastern Yunnan provinces) and northern Vietnam, at elevations between ca. 100 and 1,300 m.

Species Included and Distribution—B. arachnoidea C.I Peng, S.M. Ku & Yan Liu (China: Guangxi), B. asteropyrifolia Y.M. Shui & W.H. Chen (China: Guangxi), B. aurantiflora C.I Peng, Yan Liu & S.M. Ku (China: Guangxi), B. auritistipula Y.M. Shui & W.H. Chen (China: Guangxi), B. austroguangxiensis Y.M. Shui & W.H. Chen (China: Guangxi), B. babeana Aver. & H.Q. Nguyen (Vietnam), B. bamaensis Yan Liu & C.I Peng (China: Guangxi), B. biflora T.C. Ku (China: Yunnan; Vietnam), B. ×breviscapa C.I Peng, Yan Liu & S.M. Ku (China: Guangxi), B. cavaleriei H. Lév. (China: Guangxi, Yunnan, Guizhou; Vietnam), B. chongzuoensis Yan Liu, S.M. Ku & C.I Peng (China: Guangxi), B. cirrosa L.B. Sm. & Wassh. (China: Guangxi, Yunnan), B. crystallina Y.M. Shui & W.H. Chen (China: Yunnan), B. curvicarpa S.M. Ku, C.I Peng & Yan Liu (China: Guangxi), B. cylindrica D.R. Liang & X.X. Chen (China: Guangxi), B. daxinensis T.C. Ku (China: Guangxi), B. debaoensis C.I Peng, Yan Liu & S.M. Ku (China: Guangxi), B. fangii Y.M. Shui & C.I Peng (China: Guangxi), B. ferox C.I Peng & Yan Liu (China: Guangxi), B. filiformis Irmsch. (China: Guangxi), B. fimbribracteata Y.M. Shui & W.H. Chen (China: Guangxi), B. guangxiensis C.Y. Wu (China: Guangxi), B. huangii Y.M. Shui & W.H. Chen (China: Yunnan), B. jingxiensis D. Fang & Y.G. Wei (China: Guangxi), B. kui C.I Peng (Vietname), B. lanternaria Irmsch. (China: Guangxi; Vietnam), B. leprosa Hance (China: Guangxi, Guangdong), B. liuyanii C.I Peng, S.M. Ku & W.C. Leong (China: Guangxi), B. longgangensis C.I Peng & Yan Liu (China: Guangxi), B. longistyla Y.M. Shui & W.H. Chen (China: Yunnan), B. luochengensis S.M. Ku, C.I Peng & Yan Liu (China: Guangxi), B. luzhaiensis T.C. Ku (China: Guangxi), B. mashanica D. Fang & D.H. Qin (China: Guangxi), B. masoniana Irmsch. (China: Guangxi; Vietnam), B. morsei Irmsch. (China: Guangxi), B. nahangensis Aver. & H.Q. Nguyen (Vietnam), B. ningmingensis D. Fang, Y.G. Wei & C.I Peng (China: Guangxi), B. ningmingensis var. bella D. Fang, Y.G. Wei & C.I Peng (China: Guangxi), B. obliquifolia S.H. Huang & Y.M. Shui (China: Yunnan), B. ornithophylla Irmsch. (China: Guangxi), B. pengii S.M. Ku & Yan Liu (China: Guangxi), B. phuthoensis H.Q. Nguyen (Vietnam), B. picturata Yan Liu, S.M. Ku & C.I Peng (China: Guangxi), B. platycarpa Y.M. Shui & W.H. Chen (China: Yunnan), B. porteri H. Lév. & Vaniot (China: Guangxi, Guizhou), B. porteri var. macrorhiza Gagnep. (Vietnam), B. pseudodaxinensis S.M. Ku, Yan Liu & C.I Peng (China: Guangxi), B. pseudodryadis C.Y. Wu (China: Yunnan), B. pseudoleprosa C.I Peng, Yan Liu & S.M. Ku (China: Guangxi), B. pulvinifera C.I Peng & Yan Liu (China: Guangxi), B. retinervia D. Fang, D.H. Qin & C.I Peng (China: Guangxi), B. rhynchocarpa Y.M. Shui & W.H. Chen (China: Yunnan), B. rugosula Aver. (Vietnam), B. semiparietalis Yan Liu, S.M. Ku & C.I Peng (China: Guangxi), B. setulosopeltata C.Y. Wu (China: Guangxi), B. sinofloribunda Dorr (China: Guangxi), B. sonlaensis Aver. (Vietnam), B. subcoriacea C.I Peng, Yan Liu & S.M. Ku (China: Guangxi), B. suboblata D. Fang & D.H. Qin (China: Guangxi), B. umbraculifolia Y. Wan & B.N. Chang (China: Gaungxi), B. variifolia Y.M. Shui & W.H. Chen (China: Guangxi), B. wangii T.T. Yu (China: Guangxi, Yunnan), B. zhengyiana Y.M. Shui (China: Yunnan).

Notes—Unpublished data in our ongoing phylogenetic study indicates that B. wangii T.T. Yu, previously assigned to sect. Diploclinium (Shui et al.2002), is nested firmly within Clade SVLB and hereby transferred to sect. Coelocentrum.