Reproductive barriers are notoriously weak in Begonia. Thousands of frequently fertile Begonia hybrids have been developed artificially in cultivation (Tebbitt, 2005), a considerable number of putative natural Begonia hybrids have been observed in the field (Burt-Utley, 1985; Kiew et al., 2003; Sosef, 1994; Teo and Kiew, 1999), and hybridization and allopolyploidy seem to be common in some regional Begonia floras such as the extensively studied Begonia flora of Taiwan (Chiang et al., 2001; Ku et al., 2007; Oginuma and Peng, 2002; Peng and Chiang, 1998; Peng and Chen, 1991; Peng and Chiang, 2000; Peng and Ku, 2009; Peng and Sue, 2000). However, the genus Begonia exhibits very high numbers of local, often morphologically very distinct endemics, widespread species are rare in the genus, and population-genetic studies indicate very strong population structures and limited dispersal capabilities of some species (Hughes and Hollingsworth, 2008; Hughes et al., 2003; Matolweni et al., 2000). This may explain to a certain extent, why natural hybrids have not been reported more frequently.
Phylogenetic incongruence between independent datasets like the nuclear ITS and the non-coding plastid region employed in this study can be indicative of hybridisation (Linder and Rieseberg, 2004), but several other biological processes like incomplete homogenization of the numerous copies in nrDNA arrays, the stochasticity of lineage sorting, recombination, gene paralogy, and pseudogene formation, can result in similar patterns (Álvarez and Wendel, 2003; Feliner and Rossello, 2007; Linder and Rieseberg, 2004; Small et al., 2004). The phylogenetic analyses of the 64 taxon plastid and nuclear datasets indicate considerable phylogenetic incongruence between the plastid phylogenetic tree and the ITS phylogenetic tree. While several major clades are congruently supported, there are several taxa which show conflicting positions in the two phylogenetic trees. However, direct comparison is confounded by the poor resolution, especially of the deeper relationships, of the ITS phylogenetic tree. Major contributing factors causing this poor resolution are high levels of sequence variability and associated alignment ambiguity and high levels of homoplasy in the ITS1 and ITS2 regions. Soft incongruence, i.e. weakly supported conflicting positions in the plastid and nuclear phylogenetic trees, is probably, to a large extent, the result of these confounding factors rather than other biological processes. However, there are also several instances of hard incongruence, i.e. strongly supported conflicting positions of taxa in the phylogenetic trees, especially in the species-rich sections Platycentrum s.l. and Petermannia s.l., which need further investigation. One instance of incongruence at a deeper level of the phylogeny involves four Bornean species placed in Begonia section Petermannia. In
the cpDNA phylogenetic tree Begonia amphioxus, B. burbidgei and two unidentified species (indicated with *PET1-4 in Fig. 2.12) form a well supported clade which is not included within the strongly supported clade comprising all other species placed in section Petermannia, as well as sections Bracteibegonia and Symbegonia. In the ITS phylogenetic tree these four species are not supported as monophyletic and are nested in three different subclades in a moderately to strongly supported but poorly resolved clade comprising all samples of sections Petermannia, Bracteibegonia and Symbegonia (Fig.
2.12). Species in the two separated Petermannia clades in the cpDNA phylogenetic tree are morphologically similar: they do not have rhizomes (except for one species from Sulawesi), which is a derived condition within Clade B of the plastid phylogenetic tree, they share characteristic protogynous, two- or sometimes one-flowered female inflorescences or partial inflorescences, which are basal to or separated from the male inflorescences, and species in both groups exhibit characteristic perforate cell wall thickenings in the endothecium layer of the anthers, while the endothecium cells of species of other Asian section predominantly show U-shaped wall thickenings (Tebbitt and MacIver, 1999). The inclusion of the four orphan species in the main Petermannia clade, as indicated by the ITS data, is concordant with the morphological and anatomical data, which supports a close relationship with other species placed in section Petermannia. The plastid and ITS gene trees seem to reflect different evolutionary histories, and the observed patterns seem to be consistent with an evolutionary scenario which involves the transfer of foreign plastids in a Bornean Petermannia lineage via hybridisation, introgression and plastid capture, and subsequent diversification on Borneo. However, only Begonia amphioxus and B. burbidgei are supported as monophyletic in the ITS phylogenetic tree, while the other two species fall in two other subclades. The relationships within the Petermannia-Bracteibegonia-Symbegonia clade are only poorly resolved or poorly supported in the ITS phylogenetic tree making further interpretation difficult, but the non-monophyly of these four taxa might hint at additional confounding factors including alignment ambiguity and homoplasy, incomplete homogenization or further hybridisation and differential homogenization of the ITS copies of Bornean species in section Petermannia. A second example of incongruence within the main Petermannia clade involves the Sulawesian species Begonia capituliformis indicated with *PET5 in Fig. 2. This species is an endemic to Northern Sulawesi and falls in a well supported clade with two other Northern Sulawesian species in the cpDNA phylogenetic tree, but falls in a clade with B. rieckei and species from Southwest and central Sulawesi in the ITS phylogeny. Begonia rieckei is a fleshy-fruited species which is unusually widespread on Sulawesi, while the vast majority of Sulawesian Begonia species in section Petermannia are local endemics. The more widespread distribution of Begonia rieckei makes sympatry and hence hybridisation with other Petermannia species more likely. The observed incongruence in the plastid and nuclear DNA phylogenetic trees might indicate hybridisation, but further analyses including data from multiple accessions, data from other nuclear markers, and cloning of nrDNA regions, which was beyond the scope of this study, are needed to achieve a
72 better resolved nuclear phylogeny and to investigate whether incomplete homogenization and incompletelineage sorting were factors which contributed to the incongruence found in this group, which is likely the result of a massive, relatively recent diversification on Sulawesi in the Plio- and Pleistocene (see Chapter 3).
Further incongruence can be detected in conflicting positions of taxa placed in section Platycentrum s.l. All samples of sections Platycentrum and Sphenanthera form well supported clades in both the plastid and the ITS phylogenetic trees, but some taxa like Begonia sizemoreae and B. robusta exhibit conflicting phylogenetic position within these clades. The most conspicuous incongruence in the Platycentrum-Sphenanthera clade involves Begonia robusta, which is indicated with *SPH1 in the cpDNA and ITS phylogenetic trees in Fig. 2.12. In the analyses of the cpDNA dataset Begonia robusta falls into a clade with B. multangula, an unidentified species with strong morphological affinities to B. multangula from Sulawesi, and B. areolata. Begonia robusta and B.
multangula are morphologically most similar and are characterised by fleshy fruits and a short, stout rhizome from which well developed leafy stems arise. Hughes (2008) pointed out that some specimens are morphologically intermediate between the typical forms of these two species, and maybe of hybrid origin. Begonia areolata exhibits two-locular capsules, but a somewhat similar growth habit as B. multangula. Begonia robusta falls into a well supported clade with B. decora, B. pavonina, and B. venusta, all of which show two-locular rain-ballist capsules, in the ITS phylogenetic tree. Begonia multangula, the unidentified morphologically similar species from Sulawesi, and B. areolata are not included in this clade and can be found in two subclades within the Platycentrum-Sphenanthera clade of the ITS phylogenetic tree. Overall morphology is more concordant with the clade retrieved in the cpDNA analyses, and the separation of the morphologically most similar Begonia robusta, B. multangula, and B. aff. multangula from Sulawesi in three different clades in the ITS phylogenetic tree indicates that the ITS gene tree may not accurately reflect the species tree. A tentative somatic chromosome count of 2n = 88 for Begonia robusta (Doorenbos et al., 1998), while the majority of species in Platycentrum s.l. show a somatic chromosome of 2n = 22, indicates that allopolyploidy might have played an important role in the evolution of this group. The incorporation of xenologous loci in the nuclear genome through hybridization, and subsequent incomplete homogenization and/or different directions of homogenization to either parental species can lead to complex patterns as observed in the ITS phylogenetic tree (Álvarez and Wendel, 2003; Cronn et al., 2003; Sang et al., 1995). However, studies using multiple accessions and cloning efforts are needed to test whether different homogenization directions can be detected and whether divergent intragenomic ITS copies are present in the nrDNA arrays.
The results highlight the importance of using multiple independent sources of phylogenetic data to detect discrepancies between gene and species trees in Begonia. The considerable incongruence between the plastid and the nuclear marker phylogenetic trees
presented here may be partially explained by hybridisation. However, other biological processes, like incomplete homogenization of the numerous copies in nrDNA arrays, the stochasticity of lineage sorting, recombination, gene paralogy, and pseudogene formation, can result in similar patterns (Álvarez and Wendel, 2003; Feliner and Rossello, 2007; Linder and Rieseberg, 2004; Small et al., 2004), and further research is needed to identify and disentangle the relative impacts of these processes in causing incongruence in phylogenetic reconstructions of Begonia.
2.4.4 Conclusions
The species in the mega-diverse genus Begonia exhibit an enormous vegetative diversity and even closely related species often show conspicuous differences in growth habits, indumentum characters and leaf morphologies. It is likely that this morphological diversity arose due to both genetic drift and natural selection for adaptations to specific habitat conditions (Kidner and Umbreen, in press; Matolweni et al., 2000; Neale et al., 2006). In contrast to this, generative organs provide easily observable, qualitative and quantitative, and apparently relatively complex characters such as differences in carpel and ovary locule numbers, placentation types, and pericarp types, which have been crucial for the circumscription of sections in Asian Begonia. The results of the phylogenetic analyses and ancestral character reconstructions presented here indicate that apparently complex fruit syndromes like three- or four-locular indehiscent fruits with fleshy pericarps, and two-locular rain-ballist capsules evolved multiple times independently in Asian Begonia.
The genetic-developmental complexity, an essential criterion in character homology assessments, of the gain or loss of carpels, the inhibition of the development of locules in a three-locular ovary, the development of unilamellate or bilamellate placentae, and the development of a fleshy pericarp seem to have been overestimated in the past. The extensive homoplasy of these characters has confused systematic relationships in Asian Begonia and most Asian sections are not supported as monophyletic in the phylogenetic analyses.
The strong systematic emphasis placed on single, homoplasious characters like undivided placenta lamellae (section Reichenheimia), fleshy pericarps (section Sphenanthera), and the recognition of sections primarily based on a plesiomorphic fruit syndrome and the absence of characteristic features of other taxa (section Diploclinium) has resulted in the circumscription of several highly polyphyletic taxa. The results indicate further that the presence and absence and type of stem metamorphoses and perennating organs like tubers and rhizomes and correlated growth habits are of greater systematic value in Asian Begonia than has been assumed in the past. These vegetative characters allow us to differentiate monophyletic predominantly Malesian species groups in both sections Reichenheimia and Diploclinum from distantly related Indian, Sri Lankan and continental Asian species placed in these polyphyletic sections. The Malesian taxa are characterized by usually plagiotropically growing rhizomes, which directly produce leaves and inflorescences at their nodes, while the distantly related Indian, Sri Lankan and continental Asian species
74 placed in sections Diploclinium and Reichenheimia predominantly exhibit tubers which produce erect leafy stem, or, in acaulescent species, directly leaves and inflorescences.
Several Chinese rhizomatous species which were placed in sections Reichenheimia and Diploclinium were shown to be more closely related to the predominantly Chinese, rhizomatous section Coelocentrum, while others seem to be more closely related to section Platycentrum s.l. However, detailed comparative morphological and anatomical studies are needed to further investigate the homology and systematic importance of these organs in Begonia. Moreover, their ecological significance as perennial organs, anchor organs, and in habitat occupation and clonal reproduction are only poorly understood and studies are needed to establish whether there are correlations between precipitation, seasonality, substrate types and other environmental factors and the development of tubers, clusters of tubers and rhizomes.
Previous phylogenetic and population-genetic studies have indicated that most Begonia species seem to have low dispersal capabilities and a strong geographic structure has been observed at different geographic and taxonomic levels reaching from the continental, sectional level (Goodall-Copestake, 2005; Plana, 2003; Plana et al., 2004) to the regional, population-genetic level (Hughes and Hollingsworth, 2008; Hughes et al., 2003; Matolweni et al., 2000). The cpDNA phylogenetic trees of Asian Begonias exhibit the same trend, and indicates a stronger geographic structure than previously suggested. Clade A comprises taxa like sections Parvibegonia, and Platycentrum s.l. (incl. section Sphenanthera) and continental Asian lineages in the polyphyletic sections Diploclinium, which are most diverse on the Asian mainland, although several smaller Malesian lineages can also be recognized. Clade B comprises the predominantly Chinese section Coelocentrum, several predominantly or exclusively Malesian sections (Ridleyella, Bracteibegonia, Petermannia s.l.) and Malesian species groups in both sections Reichenheimia and Diploclinum that are only distantly related to Indian, Sri Lankan and other continental Asian species which were placed in these two highly polyphyletic sections. The strong geographic structure in cpDNA phylogeny and the general preponderance of narrow endemics in the genus highlight the potential of Begonia as model group to study the biogeography of the Southeast Asian tropical region.
The current artificial infrageneric classification of Asian Begonia has a certain diagnostic, but only poor predictive value, and has hampered the understanding of the evolution of morphological and anatomical characters, karyotypes and the ecology and biogeography of Southeast Asian Begonia. The phylogenetic trees derived from non-coding plastid data provides for the first time a reasonably resolved, supported phylogenetic framework for Asian Begonia, which has the power to inform taxonomic work, and evolutionary and biogeographical studies. This cpDNA phylogenetic framework represents an important step towards a natural re-classification of Asian Begonia, and it clarifies the phylogenetic relationships, and aspects of the character evolution and karyotype evolution within this
species-rich and morphologically diverse group. However, it also indentifies several problematic groups including Indian, Sri Lankan and continental Asian lineages of species placed in the polyphyletic sections Diploclinium and Reichenheimia, whose phylogenetic relationships are still only fragmentarily understood. Before the aim of a comprehensive, stable and natural re-classification can be achieved, it will be essential to further elucidate the phylogenetic relationships of these groups using morphological and phylogenetic studies based on a geographically robust taxon sampling and multiple independent sources of molecular data.
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