Six new species of Begonia (Begoniaceae) from limestone areas in Northern Vietnam

Background Species of Begonia are richly represented in limestone karst areas across the Sino-Vietnamese border. More than one hundred species were known, many of which were documented recently. Results In continuation of our systematic studies of Asian Begonia, we report six species of Begonia that are unknown to science, namely B. caobangensis [sect. Platycentrum], B. circularis, B. melanobullata, B. langsonensis, B.locii and B. montaniformis [sect. Coelocentrum] from Northern Vietnam. Diagnostic features that separate them from morphologically allied species are provided. Somatic chromosome numbers were determined, which supports their placement in the respective sections. Foliar SEM microphotographs were taken and described. Conclusion A careful study of the literature, herbarium specimens and living plants, both in the wild and in cultivation in the experimental greenhouse, supports the recognition of the six new species, which are described and illustrated. Electronic supplementary material The online version of this article (doi:10.1186/s40529-015-0089-3) contains supplementary material, which is available to authorized users.


Background
Begonia sect. Coelocentrum is richly represented in limestone karst areas across the Sino-Vietnamese border region, comprising more than 60 species (Peng et al. 2014a, Chung et al. 2014. Nearly half of the species in sect. Coelocentrum were discovered in the past decade (Chung et al., 2014;Peng et al. 2013Peng et al. , 2014a, mostly from southern China. The area from South China to North Vietnam harbors very high levels of biodiversity (Sodhi et al. 2004). Recently, Averyanov and Nguyen (2012) documented ten new species of Begonia from Vietnam and one new species from Laos. They also estimated the total actual number of Begonia species in eastern Indochina to be 180-200 species. It seems reasonable to speculate that there are more species of Begonia sect. Coelocentrum to be discovered in the future. In this study we report five new species of sect. Coelocentrum from Vietnam, some with very handsome maculation pattern and/or attractive leaf texture and an unusual new species of sect. Platycentrum with lanceolate and symmetric leaves. Detailed morpho logical descriptions, line drawings, color plates and foliar SEM microphotographs are provided, and chromosome numbers are reported.

Cryo scanning electron microscopy
Fresh leaves of Begonia caobangensis, B. circularis, B. melanobullata, B. langsonensis, B. locii and B. montaniformis were dissected and attached to a stub. The samples were frozen with liquid nitrogen slush, then transferred to a sample preparation chamber at −160°C and etched for 15 min at −85°C. After etching, the temperature reached −130°C for sample fracturing and coating. After coating, the samples were transferred to the SEM chamber and observed at −190°C with a cryo scanning electron microscope (FEI Quanta 200 SEM/ Quorum Cryo System PP2000TR FEI).   (Figures 1 and 2).

Distribution and ecology
Northern Vietnam, around Thang Hen Lake, Tra Linh District, Cao Bang Province ( Figure 3). Situated among limestone mountains, near the border of Vietnam and China. Growing on soil slope in evergreen broad-leaved forest, in light shade, at ca.1,000 m in elevation (Mary Sizemore, pers. comm.). Begonia caobangensis occurs also in Tuyen Quang province (Trinh Ngoc Bon, pers. comm.), which is adjacent to the southwest border of Cao Bang.

Etymology
The specific epithet refers to the locality of the type collection.

Distribution and ecology
Endemic to Thach An District, Cao Bang Province, Vietnam ( Figure 3), near the border between Vietnam and China. Growing in cracks of mossy rocks on semi-shaded limestone cliffs in evergreen broad-leaved forest, elevation ca. 600 m.

Etymology
The specific epithet refers to the circular foliar variegation of the new species.

Distribution and ecology
Endemic to Thach An District, Cao Bang Province, Vietnam (Figure 3), near the border between Vietnam and China. On semi-shaded wet cliffs or steep limestone slopes in evergreen broad-leaved forest, elevation at 300-400 m.

Etymology
The specific epithet refers to the color of the bullae on leaf surface.  (Figures 8 and 9).

Distribution and ecology
Endemic to Huu Lung Protected Area, Huu Lien District, Lang Son Province, Vietnam (Figure 3). On semishady cliff face or steep limestone slopes in evergreen broad-leaved forest, elevation ca. 125-165 m.

Etymology
The specific epithet refers to Lang Son Province, where the new species was discovered.

Notes
Begonia langsonensis somewhat resembles B. ornithophylla Irmsch. (southwest Guangxi, China) in leaf shape and aspect. However, B. langsonensis is nearly glabrous throughout, whereas B. ornithophylla has sericeous petiole, puberulous abaxial blade, as well as velutinous to villous pedicel and peduncle. In addition, B. langsonensis differs from the latter in the bracts widely to very widely ovate (vs. ovate-triangular) as well as the primary and secondary veins on leaf adaxial surface elevated (vs. sunken).  (Figures 10 and 11).

Distribution and ecology
Begonia locii is endemic to Huu Lung Protected Area, Huu Lien District, Lang Son Province, Vietnam (Figure 3), occurring on semi-shaded limestone rock face, elevation 125-165 m.

Etymology
Begonia locii is named in honor of Professor Phan Ke Loc for his contribution to Vietnamese botany.

Notes
Begonia locii resembles B. luochengensis S.M. Ku, C.-I Peng & Yan Liu from Guangxi, China in leaf shape and maculation pattern, but is clearly distinct from the latter in the percurrent and spiderweb-like leaf venation (vs. pinnate) near the attachment of the petioles; upper leaf surface densely covered with small raised cones (vs. nearly flat); and hirsute (vs. glabrous) ovaries. The new species is also similar to B. pengii S. M. Ku & Yan Liu in leaf maculation pattern, vestiture, and hirsute ovaries, differing by the leaf basifixed (vs. peltate); bract margin denticulate and glandular (vs. denticulate and ciliate). A comparison of salient characters of the three species, all of sect. Coelocentrum, is shown in Table 4.  (Figures 12 and 13).

Etymology
The specific epithet refers to the leaf surface with dense conic bullae, which is reminiscent of the peak clusters in Sino-Vietnamese limestone karst landform.  Nguyen, another bullate-leaved species recently documented from north Vietnam (Averyanov & Nguyen 2012), differing in the leaves widely ovate to widely elliptic (vs. broadly oblique-ovate or oblique-reniform), apex caudate (vs. obtuse) and each conic bullae 2-4-tipped (vs. 1-tipped).

Notes
A detailed comparison of the new species with B. ferox and B. nahangensis are provided in Table 5.

Chromosome cytology
1. Begonia caobangensis (Peng 23895), 2n = 22, Figure 14A The 22 chromosomes, ranging from ca. 1.1 to 1.8 μm long, show a gradual change in length. Most chromosomes had centromeres at the median/submedian positions. No satellite chromosomes were observed. (Peng 22610), 2n = 30, Figure 14B Among the 30 chromosomes, the first pair of median chromosomes (ca. 2.1 μm long) was relatively longer than the rest and the remaining 28 gradually varied from ca. 1.2 to 1.9 μm long. Except for the shorter chromosomes, most chromosomes had centromeres at the median/submedian positions. No satellite chromosomes were observed.

5.
Begonia locii (Peng 21943), 2n = 30, Figure 14E The 30 chromosomes gradually varied from ca. 0.9 to 1.7 μm long. Almost all chromosomes had centromeres at median/submedian positions. Satellites were observed at distal regions of short arms in the second pair of the longest median chromosomes.   The holotype (Peng 24609) and one of the paratypes (Peng 24613) had 2n = 30, which is the prevalent chromosome number for members of sect. Coelocentrum. However, another collection (Peng 24610) showed aneuploidy with 2n = 30, 31, 32, 33 in different individuals. The aneuploids shared chromosomal features in common in the respective chromosome complements: The first two chromosomes were relatively longer, about 1.8-2.0 μm (arrows in Figures A-D; Nos. 1, 2 in Figures  E-H), and the rest were shorter, about 1.0-1.5 μm long. The two longest chromosomes were clearly metacentric, however, the centromere positions of most other chromosomes could not be determined. Satellites were not observed.

Conclusion
A careful study of the literature, herbarium specimens and living plants, both in the wild and in cultivation in the experimental greenhouse, supports the recognition of the six new species, which are described and illustrated. Our study also suggests that there are more new species in limestone karst areas across the Sino-Vietnamese border region to be discovered in the future.