Begonia myanmarica (Begoniaceae), a new species from Myanmar, and molecular phylogenetics of Begonia sect. Monopteron

Background A new species, Begonia myanmarica, was discovered from Myanmar and herein documented. Characterized by a single developed wing in the ovary/fruit, this species would be assigned to sect. Monopteron (sensu Doorenbos et al. in The sections of Begonia including descriptions, keys and species lists: studies in Begoniaceae VI. Wageningen Agricultural University, Wageningen, 1998) that is known by B. griffithiana and B. nepalensis from the Himalaya. To confirm its sectional assignment, we conducted morphological, phylogenetic and cytological studies. Results Morphological observations indicated that B. myanmarica was distinguishable from the two known species of sect. Monopteron by the leaf shape and size, 1-locular ovary, parietal placentation and chromosome number. Molecular phylogenetic analysis using nrITS sequences showed that B. myanmarica was not allied with the clade of sect. Monopteron, though both were nested within sect. Platycentrum-sect. Sphenanthera clade. Conclusions Studies of morphology, molecular phylogenetics and cytology support the recognition of the new species, Begonia myanmarica, which is fully described and illustrated. Our results also indicate that B. myanmarica is not closely related to species previously assigned to sect. Monopteron, suggesting that the fruit morphology of a single developed wing in the ovary/fruit characterizing sect. Monopteron is homoplasious.


Background
, comprising more than 1800 species classified into 68 sections (Doorenbos et al. 1998;Hughes et al. 2015;Christenhusz and Byng 2016), is one of the largest genera of vascular plants. With more than 760 Begonia species in Asia, Doorenbos et al. (1998) Forrest & Hollingsw.] (Ku 1999;Shui et al. 2002;Forrest and Hollingsworth 2003;Ku et al. 2007). These 22 Asian sections are highly unequal in species numbers: eight of the large sections (Petermannia, Platycentrum, Diploclinium, Reichenheimia, Coleocentrum, Parvibegonia, Sphenanthera, and Symbegonia) comprise 95% of Asian Begonia species and the rest 14 sections each with less than five species . Several molecular phylogenetic studies have demonstrated the paraphyly or polyphyly of these large sections, suggesting homoplasy of morphological characters used for current sectional delimitations (Tebbitt et al. 2006;Thomas et al. 2011;Chung et al. 2014). However, few studies have tested the monophyly of small Asian section of Begonia thus far [but see Rajbhandary (2010); Rubite (2010); ].
Myanmar is botanically a most interesting country, but there have been no critical floristic surveys for nearly half a century. Thus far about 60 species of Begonia have been recorded from Myanmar (Hughes 2008;Tanaka and Hughes 2007;Tanaka and Hayami 2011;Peng et al. 2014b;Tanaka and Peng 2016). During the fieldwork in western Myanmar on 2 February 2012, the second author (YDK) collected an unknown Begonia with only one developed wing in ovary/fruit, which is the key character of Begonia sect. Monopteron sensu Doorenbos et al. (1998) first delimited by de Candolle (1864) Doorenbos et al. 1998). Begonia nepalensis, the type species of sect. Monopteron, is native to Bhutan, Nepal and India (Fig. 5;Doorenbos et al. 1998;Hughes et al. 2015). Its chromosome number was reported to be 2n = 16 (Legro and Doorenbos 1971), with an uncertain chromosome count 2n = 28-42 by Sharma and Bhattacharyya (1961). Begonia griffithiana, occurring in Bhutan and India (Fig. 5), is characterized by lanceolate to oblong leaves with subcordate base. Chromosome number of B. griffithiana was documented as 2n = 22 (Doorenbos et al. 1998). Based on recent systematics and phylogenetics of Begonia, sect. Monopteron is nested within the Platycentrum-Sphenanthera clade (Rubite 2010;Rajbhandary et al. 2011;Leong 2017).
Although morphology of the 1-winged ovary/capsule of the undescribed Begonia should be assigned to sect. Monopteron, it differs from B. griffithiana and B. nepalensis significantly the leaf shape, leaf size and distribution. In this study, we described it as a new species. We also provide detailed morphological data and molecular phylogenetic analysis to elucidate the sectional assignment for this species.

Morphological observations
Rhizomes of Begonia myanmarica collected by YDK from Myanmar were cultivated in the experimental greenhouse of the Biodiversity Research Center, Academia Sinica, Taipei, Taiwan. Fully grown plants with flowers and fruits (Peng 23565, 23566) were used for morphological observation. The two species of sect. (Peng 20851) and B. nepalensis (Peng 20854), cultivated in the greenhouse were also studied as a comparison.

Chromosome preparations
Root tips were obtained from cultivated materials from greenhouse of Academic Sinica. Somatic chromosome of the new species, B. myanmarica (Peng 23566), and two species of sect. Monopteron: B. griffithiana (Peng 20851) and B. nepalensis (Peng 20854), were examined using root tips following the methods by Peng et al. (2014a).

Phylogenetic analyses
DNA sequences of the nuclear ribosomal internal transcribed spacer (nrITS) were used to evaluate the phylogenetic relationship among new species and the two species of sect. Monopteron. DNA extraction, PCR amplification and DNA sequencing followed Chung et al. (2014). To test the monophyly of sect. Monopteron and sectional assignment of new species, nrITS of 96 species used in Chung et al. (2014) were adopted for phylogenetic analysis (see Appendix for details). Alignment was conducted using MUSCLE implemented in MEGA5.2 (Tamura et al. 2011) and verified in Mesquite v3.03 (Maddison and Maddison 2015). Phylogenetic relationships were constructed by Bayesian Inference (BI) method. The best nucleotide substitution models were determined by Modeltest v2.7 (Posada and Crandall 1998). For BI analysis, the consensus topology was based on Markov chains algorithm implemented in MRBAYES 3.0b4 (Huelsenbeck and Ronquist 2001). Four chains of Markov chain Monte Carlo (MCMC) simulation were carried out for 1,500,000 generations each with trees sampled per 500 generations. The first 500 trees of sampled trees were discarded before the node probability was calculated (posterior probability: PP).

Distribution and habit
Known only from the type locality.

Etymology
The epithet refers to Myanmar (formerly Burma) where it was discovered.

Chromosome cytology
Somatic chromosome at metaphase of B. myanmarica were shown to be 2n = 38 in this study (Fig. 4c).

Discussion
Begonia myanmarica has only one developed wing in ovary/fruit (Figs. 2k, 3c, d), the key character of sect. Monopteron in Begonia (Doorenbos et al. 1998). The new species, however, deviates from sect. Monopteron with axillary placentation and two locules in ovary (Fig. 3e, f ) in having 1-locular ovary and parietal placentation (Fig. 2j). Additionally, B. myanmarica has ovate to broadly ovate leaves and large leaves (ca. 20-40 cm long, 22-30 mm across) (Fig. 2b, c), whereas leaves of B. griffithiana and B. nepalensis are lanceolate to oblong and no longer than 20 × 10 cm (Fig. 3a, b). Cytologically,  (Fig. 6), suggesting that the fruit morphology of a single developed wing in the ovary/fruit is homoplasious. Morphologically, the key characters for sect. Platycentrum-sect. Sphenanthera clade are evergreen, rhizomatous and two locules in ovary (Leong 2017). Begonia myanmarica is evergreen with stout rhizome, but having 1-locular ovary. Compared with other species in sect. Platycentrum-sect. Sphenanthera, B. myanmarica is unique with a single developed wing and having 1-locular ovary not known in any other taxa in this clade. Further studies with increasing sampling of Myanmar Begonia are needed to place B. myanmarica in its proper infrageneric position.

Conclusion
Studies of morphology, molecular phylogenetics and cytology support the recognition of the new species, Begonia myanmarica, which is fully described and illustrated. Our results also indicate that B. myanmarica is not closely related to species previously assigned to sect.  (Hughes et al. 2015) Monopteron, suggesting that the fruit morphology of a single developed wing in the ovary/fruit characterizing sect. Monopteron is homoplasious.
Authors' contributions YDK, KMH, and SHC conducted the fieldwork and collected the new species from Myanmar; YDK and CIP took color photographs of B. myanmarica from the wild and the experimental greenhouse respectively; YHT carried out the morphological observation and undertook laboratory analyses; YK carried out the cytological study; YHT, CIP and KFC prepared the manuscript. All authors read and approved the final manuscript.   Chung et al. (2014)