Comparative phylogenetic analyses of limestone and non-limestone Begonias in Peninsular Malaysia

Karsts of Southeast Asia cover ~400,000 km2, with tropical limestone karsts possessing highly biodiverse flora in many, varied microhabitats that are unique from lowland- and hill forests. Begonia L. is a pantropical mega-genus with ~1,550 described species to date, ideal for studying evolutionary patterns and factors driving tropical population- diversity and speciation. Begonias grow on multiple substrates including limestone and granite, sandstone, quartzite, on steep earth slopes and near streams in primary forests. Niche partitioning is commonly observed, resulting in many single-site endemic Begonia species especially on limestone karsts. Molecular phylogenetic analyses were performed on combined chloroplast ndhF-rpl32 and nuclear internal transcribed spacer ITS sequences that included 30 selected Begonia species representing all sections from Peninsular Malaysia (Pen. Msia; ten limestone-, thirteen forest- and seven granite species). Results of Bayesian inference and molecular dating showed evolution of Begonia from continental Asia into Pen. Msia during the mid-late Miocene period of ~ 10.65 mya (9.7 - 12.12 mya), with at least two independent dispersal events observed by the early splitting of the ancestral lineage into two major clades i.e. clades A- and B at 9.85 mya (HPD 9.7-10.36 mya) and 8.85 mya (HPD 7.7-11.23 mya) respectively; followed by multiple speciation events colonizing limestone- as well as non-limestone (forest, granite) habitats. Begonia speciation into limestone habitats occurred three times, with climate indicated as more important factor than substrate in diversification - clade A contained the Indian/Continental Asia (monsoon climate) species, i.e. Sect. Platycentrum and Parvibegonia, resolved slightly older than clade B where Sect. Petermannia, Jackia and Ridleyella formed a Sunda Shelf (equatorial climate) centred group. Strong population structuring by locality, common in Begonias, was also observed (e.g. B. kingiana, B. foxworthyi, B. nurii, B. sinuata), regardless of distribution. However, for widespread species (B. kingiana, B. sinuata) behavioural (physiological) inertia on niche evolution, and/or genome size variation and dynamics (genome evolution) may potentially explain species cohesion. Phylogenetics relationships are mostly concordant with current sectional classifications excepting Sect. Parvibegonia, Sect. Diploclinium and two paraphyletic species complexes. Conservation of Begonias and biodiverse limestone karsts in Malaysia remains concerning due to ongoing threats (large-scale quarrying/mining, human encroachment), and most are still without any legal protection.