**5.4 Polyploidisation**

Polyploidisation within *Begonia* may play a role in the diversification of lineages, the formation of new species, and morphological innovation. Evidence for the importance of polyploidy in *Begonia* evolution comes from the wide range of chromosome number, even in related species. Polyploids can arise by means of somatic mutations in meristematic cells or unreduced (2n) gametes (Bretagnolle & Thompson, 1995; Otto & Whitton, 2000). Harlan & De Wet (1975) showed that many plant species produce 2n gametes, and it is now generally accepted that 2n gametes are the driving force behind the formation of polyploids in nature (Bretagnolle & Thompson, 1995). The occurrence of 2n gametes in a *Begonia* collection has been described by Dewitte et al. (2009b), where 10 of the 70 investigated genotypes (collections of both species and cultivars) produced 2n pollen. The unreduced pollen grains were able to germinate and could be used to produce progeny. The occurrence of 2n egg cells was not investigated, but ploidy analysis of the progeny proved that 2n egg cells do

The Origin of Diversity in *Begonia*:

detailed studies on this topic are required.

maintaining the distinct identities of *Begonia* species.

evidence is required.

**6. Hybridisation** 

Genome Dynamism, Population Processes and Phylogenetic Patterns 39

the presence of paracentric inversions during crossing-over (Newman, 1966). Paracentric inversion loops are crossing over configurations that result in abnormal meiotic end products such as dicentric chromosomes (with 2 centromeres) and acentric (without centromere) fragments. Consequently, new chromosome constitutions may arise during meiosis. Furthermore, chromosome inversions may affect rates of adaptation and speciation because it promotes reproductive isolation of species (Noor et al., 2001; Hoffmann & Rieseberg, 2008). However, the importance of inversions in adaptive evolution has rarely

Univalent formation was frequently observed in *Begonia* (Dewitte et al., 2010; Okuna & Nagai, 1953). The presence of these univalents resulted in lagging chromosomes: chromosomes with a delayed movement to the poles. In some cases (dependent on the hybrid), univalents did not migrate to the poles but formed micronuclei. Although this usually leads to unbalanced chromosome segregation and sterility (Bretagnolle & Thompson, 1995), most of the studied hybrids were fertile. It is uncertain to what degree *Begonia* are 'buffered' against unbalanced chromosome segregation. If the resulting aneuploid gametes are fertile, this process may influence the chromosome number transmitted through the progeny and the genome stabilisation process. However, more

In general, cytological studies (mainly in artificial hybrids) have shown that chromosome behaviour during meiosis is very dynamic which may have important consequences for chromosome evolution within the genus. However, further clarification with molecular

Hybridisation and polyploidisation have played a major role in the evolution of plant species, and the stabilisation of hybrid lineages may contribute to species diversity (Mallet, 2007; Paun et al., 2009; Rieseberg & Carney, 1998). However, estimating the frequency of hybrid speciation is difficult, as evidence that hybridisation has let to speciation is hard to obtain, particularly in homoploid hybrids (hybrids without a change in ploidy level) (Mallet, 2007). The role that hybridisation has played in contributing new lineages in diverse tropical biomes has yet to be addressed in detail (with some exceptions such as neotropical orchids, eg Pinheiro et al., 2010), however hybrid speciation may be one of the factors contributing to species diversity in large genera such as *Begonia*. Support for hybridisation and introgression being a mechanism for the generation of diversity would require an understanding of the strength of reproductive isolation between parental species, including factors such as the fertility of hybrids, and few studies have focused on this in *Begonia*. Several natural *Begonia* hybrids have been described including: *B. x breviscapa*, *B. x chungii* and *B. x taipeiensis* (Peng & Ku, 2009; Peng & Sue, 2000; Peng et al., 2010). This illustrates weak reproductive barriers between *Begonia* species that co-occur in the wild, which is typical of a genus that has been widely exploited in the development of horticultural varieties (Tebbitt, 2005). However, in most cases the number of species that co-occur at a single locality in *Begonia* is low. The ease of hybridisation under experimental conditions, combined with the low frequency of hybrid occurrence in the wild, indicates that habitat specialisation and non-overlapping geographic distributions may be important in

been addressed and therefore its role in adaptive shifts is not yet clear.

occur (Dewitte et al., 2010a). Unreduced egg cells have also been observed in the hybrid *B. cucullata* x *B. schmidtiana* during breeding of semperflorens *Begonia* (Dewitte et al., 2010a; Horn, 2004).

Unreduced gametes have been shown to occur in species as well as hybrids (Dewitte et al., 2010a; Dewitte et al., 2009b). This suggests natural polyploid lineages may occur at a low frequency in the wild. However, hybrids may produce 2n gametes at c. 50 times higher frequency than species (Ramsey & Schemske, 1998). In horticulture, interspecific hybrids are often a source of 2n gametes. These 2n gametes can transfer high levels of heterozygosity (Bretagnolle & Thompson, 1995; Ramanna & Jacobsen, 2003), promoting polyploid establishment, and in some cases 2n gametes may be the only viable gametes in hybrids (Barba-Gonzalez et al., 2004; Dewitte et al., 2010b).

### **5.5 Genome size variation**

The combined effect of polyploidy and genome stabilisation is a highly variable genome size across the genus. By using flow cytometry, Dewitte et al. (2009a) observed variation in genome size between 1C = 0.23 pg and 1.46 pg, but genome size did not positively correlate with chromosome number. The largest known discrepancies between genome size and chromosome number were observed in *B. pearcei* and *B. boliviensis,* which have the largest genome size measured in *Begonia* to date *(*1C = 1.46 pg*)*, although they contain the lowest chromosome number of all genotypes analysed (2n = 26 and 28, respectively). The lack of correlation between genome size and chromosome number can be explained by the strong differences in chromosome size. Dewitte et al. (2009a) showed that the total chromosome volume in a cell was positively correlated to the genome size. Smaller chromosomes contribute less to the genome size than large chromosome do, even if the number of chromosomes is equal.

### **5.6 Meiosis**

Chromosome pairing at metaphase I can indicate whether plants are diploids or secondary polyploids (autopolyploid or allopolyploid), but few cytological studies have focused on meiosis in *Begonia*. In *B. evansiana* (Okuna & Nagai, 1953) and *B. x chungii* (Peng & Ku, 2009), several loosely associated bivalents (paired chromosomes), which almost resemble univalents (unpaired chromosomes), were observed during chromosome pairing. Furthermore, many secondary associations between bivalents were observed in *B. evansiana*  (Okuna & Nagai, 1953), consistent with observations in *B. semperflorens* and *B. rex* (Matsuura & Okuno, 1943). These observations suggested *B. evansiana* is a secondary polyploid, derived from two species with X=6 and 7 as their basic chromosome numbers.

In horticultural crosses, the interspecific hybrid *B*. 'Tamo' (2n = 23) showed irregular chromosome pairing with multivalents of up to 7 chromosomes (Dewitte et al., 2010c). These associations were often observed between chromosomes with large differences in size, which indicated associations between non-homologous chromosomes and possible recombination between these non-homologous DNA segments (illegitimate recombination). Illegitimate recombination is the driving force behind genome size decrease in *Arabidopsis* (Bennetzen et al., 2005; Devos et al., 2002), but can also be an important mechanism for exon shuffling, a major process for generating new genes (Long, 2001; Long et al., 2003).

In some hybrids, tight associations between chromosomes resulted in chromosome bridges and fragments during anaphase I (Dewitte et al., 2010c). This might be indirect evidence for the presence of paracentric inversions during crossing-over (Newman, 1966). Paracentric inversion loops are crossing over configurations that result in abnormal meiotic end products such as dicentric chromosomes (with 2 centromeres) and acentric (without centromere) fragments. Consequently, new chromosome constitutions may arise during meiosis. Furthermore, chromosome inversions may affect rates of adaptation and speciation because it promotes reproductive isolation of species (Noor et al., 2001; Hoffmann & Rieseberg, 2008). However, the importance of inversions in adaptive evolution has rarely been addressed and therefore its role in adaptive shifts is not yet clear.

Univalent formation was frequently observed in *Begonia* (Dewitte et al., 2010; Okuna & Nagai, 1953). The presence of these univalents resulted in lagging chromosomes: chromosomes with a delayed movement to the poles. In some cases (dependent on the hybrid), univalents did not migrate to the poles but formed micronuclei. Although this usually leads to unbalanced chromosome segregation and sterility (Bretagnolle & Thompson, 1995), most of the studied hybrids were fertile. It is uncertain to what degree *Begonia* are 'buffered' against unbalanced chromosome segregation. If the resulting aneuploid gametes are fertile, this process may influence the chromosome number transmitted through the progeny and the genome stabilisation process. However, more detailed studies on this topic are required.

In general, cytological studies (mainly in artificial hybrids) have shown that chromosome behaviour during meiosis is very dynamic which may have important consequences for chromosome evolution within the genus. However, further clarification with molecular evidence is required.
