**5.1 Chromosome number**

34 The Dynamical Processes of Biodiversity – Case Studies of Evolution and Spatial Distribution

Kwazulu-Natal, and population structure assessed using microsatellite markers. A similar population structure was observed to *B. dregei* and *B. homonyma*. Levels of population differentiation were high and there was significant differentiation between populations, even within subpopulations at a small spatial scale. The genetically isolated nature of *B. sutherlandii* populations suggest effective interpopulation dispersal is rare. There was no significant link between genetic and geographic distance suggesting that this differentiation

A third study of population structure in *Begonia socotrana* and *B. samhaensis* at the Socotra archipelago also shows the same pattern of strongly isolated populations (Hughes et al., 2003). The low intraspecific gene flow could be due to both poor seed dispersal in the sheltered conditions of the forest floor and to limited pollen flow. *Begonia* flowers do not attract specialist pollinators but practice deceit pollination by generalist pollinators such as small bees and flies. Analysis of this pollination mechanism in the wild has confirmed that it does

Isolation-by distance may contribute to speciation in the genus. As described by Hughes & Hollingsworth (2008), the population-level data are congruent with the macro-evolutionary patterns observed in the genus. Molecular phylogenies confirm that *Begonia* is characterized by geographically constrained monophyly, species with narrow geographical ranges, very few widespread species, and high levels of morphological differentiation between

The limited dispersal of many *Begonia* makes them a useful group to search for the biogeographic signature of refugia (Sosef, 1994). Refugia are regions where vulnerable lineages could survive periods of dramatic climate change, such as ice ages. When the climate becomes favorable again, species disperse outwards from the refugia. This generates a distinctive pattern in the geographic distribution of genetic diversity, with former refugia having greater genetic diversity. The poor dispersal of *Begonia* means regions with high numbers of *Begonia* species are possible former refugia. Species of the African sections *Loasibegonia* and *Scutobegonia* are restricted to humid and shady locations and are proposed to have been especially sensitive to the period of Pleistocene climate oscillations, and dispersed slowly afterwards (Sosef, 1994). Centers of diversity for these sections are situated in West and Central Africa. Plana et al. (2004) also used *Begonia* diversity to identify the island of Sao Tome as a pre-Pleistocene refuge, and suggested different mainland areas in West Africa as refuges. It is quite possible that a number of different refugia existed for

*Begonia* exhibit a highly dynamic genome, with large variation in chromosome number, genome size and mean chromosome size as well as divergent chromosome structure, even between closely related species (Dewitte et al., 2009a). Traditionally, investigations at the chromosome level have been hindered by the small size of *Begonia* chromosomes, the difficulty in visualising centromeres, and few reliable karyograms. However, recent investigations using horticultural hybrids between divergent species, as well as cytological studies in the context of phylogenetic relationships, have greatly improved our

is caused by genetic drift rather than through long-term isolation.

results in a low seed set (Ågren & Schemske, 1993; de Lange & Bouman, 1999).

populations of the few widespread species (Hughes & Hollingsworth, 2008).

**5. Cytological investigations and genome size comparisons** 

**4.2 Refuge** *Begonia*

lineages with different ecological tolerances.

understanding of this cytologically interesting genus.

The occurrence of particular chromosome numbers in a given group is important for predicting reproductive barriers between species and the potential fertility of the hybrids, and can be indicative of a close evolutionary relationship. Among *Begonia* species, chromosome numbers range from 2n = 16 for *B. rex* to 2n = 156 for *B. acutifolia*. Between these extremes, a wide range of chromosome numbers have been described (Doorenbos et al., 1998; Legro and Doorenbos, 1969; Legro and Doorenbos, 1971; Legro and Doorenbos, 1973). Many species or cultivars exhibit chromosome numbers of 2n = 26 or 28 (x = 13 or 14) or a multiple of this number. Within the horticultural tuberous begonia group, derived from interspecific crosses between American *Begonia*, chromosome numbers of 2n=27,28 (diploid), 41,42 (triploid) and between 52 and 56 (tetraploid) are most common (Legro and Haegeman, 1971; Haegeman, 1979), but variation outside this sequence exists. In Asian *Begonia*, 2n = 22 (x = 11) is the most frequently observed chromosome number. A phylogeny of non-coding cpDNA also indicates a base chromosome number of x = 15 may be ancestral within Asian *Begonia*, with chromosome counts of 30 or 44 as diploid and triploid derivatives (Thomas, 2010; Thomas et al., in press).

The search for a basic chromosome number is complex as there is no common number observed in the group, even taking into account the prevalence of polyploidy in the horticutural varieties assayed. Some authors (Matsuura & Okuno, 1936; Matsuura & Okuno, 1943; Okuna & Nagai, 1953; Okuna & Nagai, 1954) have suggested x = 6, x = 7 and x = 13 as the basic chromosome number, where x=13 may be of secondary origin. By using genomic *in situ* hybridisation (GISH), Marasek-Ciolakowska (2010) concluded that x = 7 may be the basic chromosome number of *B. socotrana*. They based this conclusion on the presence of 7 *B. socotrana* chromosomes and 56 chromosomes derived from tuberous *Begonia* in Elatior hybrids. An alternative explanation of the genomic composition of these Elatior hybrids is selective chromosome elimination of *B. socotrana* chromosomes after hybridisation. Selective chromosome elimination is a genome stabilisation process, and cytological investigation by Arends (1970) supports a role for it in the breeding of Elatior *Begonia*, observing 9 or 12 *B. socotrana* chromosomes in some Elatior hybrids.

The inferred African origin for *Begonia* may suggest a basic chromosome number will be found in these taxa, especially early branching lineages. However it is uncertain how the genomic composition, particularly in terms of chromosome number, has changed in extant African species relative to their ancestors. Most of the described chromosome numbers in African taxa vary between 36 and 38, but counts of 22, 26 and 28 have also been made. Chromosome numbers of 22, 26 and 28 appear to be prevalent in the East-African seasonally adapted *Begonia* from the sections *Rostrobegonia* and *Sexalaria,* which diverged very early during *Begonia* evolution, and from the sections *Augustia* and *Peltaugustia.* These sections show a closer relationship to American and Asian sections than to other African sections.

The closely related *Hillebrandia sandwichensis* has a chromosome number of 2n = 48 (Kapoor, 1966), probably the result of a polyploidisation of a 'diploid' with 2n = 24. Within the most related family Datiscaceae, 2n = 22 is reported for *Datisca cannabina* (Gupta et al., 2009). The chromosome numbers within the family Cucurbitaceae are very diverse, but many species posses chromosome numbers between 2n = 20 and 26 or multiples of these numbers, and 2n = 14, 16 and 18 are also widely reported. In the Coriariaceae, multiples of 20 were observed (2n = 20, 40 and 60) while in Corynocarpus, 2n = 44 or 46 is reported. An exact list of chromosome numbers and references in the abovementioned families is available at the TROPICOS® database (www.tropicos.org). These numbers indicate that basic chromosome

The Origin of Diversity in *Begonia*:

**5.4 Polyploidisation** 

*B. socotrana* and large *B. x tuberhybrida* chromosomes.

transposable elements and their defunct remnants.

Genome Dynamism, Population Processes and Phylogenetic Patterns 37

observed in horticultural hybrids, including Elatior hybrids that are characterized by small

Apart from *B. pearcei* and *B. boliviensis*, South American species possess smaller chromosomes than Asian, African and Middle American species (Dewitte et al., 2009a). Different mechanisms, such as transposon activity, multiple deletions or other genetic rearrangements may have played an important role in the generation of this chromosome size variation (Devos et al., 2002; Kubis et al., 1998; Sanmiguel & Bennetzen, 1998). Bennetzen (2002) showed that more than 60% of some plant genomes consist of

Fig. 3. Prometaphase mitotic chromosome spread of *B*. 'Tamo' (2n = 23), stained with DAPI.

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

Intense fluorescence signal indicates satellites (Dewitte et al., 2009a). Bar = 5 µm.

numbers within these families are situated between x = 10 and 13. However, within the basally branching family Anisophylleaceae, chromosome numbers of x = 7 and x = 8 are reported (Tobe & Raven, 1987).

The above data suggest a diploid chromosome number for the genus *Begonia* between 20 and 26; chromosome numbers of 2n = 36 or 38 were probably established after polyploidisation and genome stabilisation early in the evolution of this genus. Given that the 'older' sections within the genus have a chromosome number of 2n = 26, we suggest that x = 13 is the original basic chromosome number, a chromosome number observed among sections across the world. However, it cannot be excluded that the basic chromosome number of x = 13 is of secondary origin and arose from a fusion between cells with x = 6 and 7 (as suggested by meiotic studies on *B. evansiana* by Okuna & Nagai (1953)), although karyomorphological studies in *Begonia* do not support x = 6 or 7 as basic chromosome number. If this was the case, the hybrid B276 (2n = 50; Dewitte et al., 2009a) would be a near octoploid and 8 homoeologous chromosomes should be identified, instead of the near tetraploid spread observed (x = 14). Further evidence from Oginuma & Peng (2002) show *B. palmata* and *B. aptera* (both 2n = 22) have 2 secondary constriction chromosomes, which supports a basal number of x = 11 in Taiwanese *Begonia*.

Subsequently, chromosome numbers have diverged resulting in some 'new' basic chromosome numbers for some subgroups (eg x = 11 or x = 14), through selective chromosome elimination and polyploidy. Other derived numbers may have resulted from interspecific hybridisation, and Legro & Doorenbos (1969) suggested that 2n = 22 possibly originated out of a cross between 2n = 16 and 2n = 28.

#### **5.2 Chromosome structure**

Few studies have looked into changes in chromosome structure during *Begonia* evolution. Although karyomorphological data in *Begonia* are limited, many secondary constriction (SC) chromosomes have been detected. A SC chromosome consists of a satellite connected to the main body by the secondary constriction, a thin strip of the chromosome (Fig. 3). Oginuma & Peng (2002) showed that up to 63% of the chromosomes in a cell can be secondary or tertiary constriction chromosomes. Moreover, all 14 species of Taiwanese *Begonia* (including *B. palmata* and *B. aptera* with 2n = 22) surveyed possessed SC, except for *B. fenicis* (2n = 26), where the exact position of centromeres could not be determined for some chromosomes. Dewitte et al. (2009a) observed SC chromosomes in 6 out of 11 genotypes investigated. During prophase, the satellites were so loosely associated to their main body that they could easily be misidentified as separate chromosomes. Additional evidence for SC chromosomes in *Begonia* was presented by Legro & Doorenbos (1969).

These data suggest that after polyploidisation, chromosome translocations occur, which are followed by a decrease in chromosome number and genome stabilisation (Oginuma & Peng, 2002).

#### **5.3 Chromosome size**

Flow cytometric measurements of genome size, combined with chromosome counts, revealed a 12-fold difference in the mean chromosome size between the species *B. dietrichiana* and *B. pearcei* (Dewitte et al., 2009a). Moreover, as only 15 of the 66 sections of the genus *Begonia* were involved in this study it is likely that greater differences will be found when other sections are surveyed. Differences in chromosome size have also been

numbers within these families are situated between x = 10 and 13. However, within the basally branching family Anisophylleaceae, chromosome numbers of x = 7 and x = 8 are

The above data suggest a diploid chromosome number for the genus *Begonia* between 20 and 26; chromosome numbers of 2n = 36 or 38 were probably established after polyploidisation and genome stabilisation early in the evolution of this genus. Given that the 'older' sections within the genus have a chromosome number of 2n = 26, we suggest that x = 13 is the original basic chromosome number, a chromosome number observed among sections across the world. However, it cannot be excluded that the basic chromosome number of x = 13 is of secondary origin and arose from a fusion between cells with x = 6 and 7 (as suggested by meiotic studies on *B. evansiana* by Okuna & Nagai (1953)), although karyomorphological studies in *Begonia* do not support x = 6 or 7 as basic chromosome number. If this was the case, the hybrid B276 (2n = 50; Dewitte et al., 2009a) would be a near octoploid and 8 homoeologous chromosomes should be identified, instead of the near tetraploid spread observed (x = 14). Further evidence from Oginuma & Peng (2002) show *B. palmata* and *B. aptera* (both 2n = 22) have 2 secondary constriction chromosomes, which

Subsequently, chromosome numbers have diverged resulting in some 'new' basic chromosome numbers for some subgroups (eg x = 11 or x = 14), through selective chromosome elimination and polyploidy. Other derived numbers may have resulted from interspecific hybridisation, and Legro & Doorenbos (1969) suggested that 2n = 22 possibly

Few studies have looked into changes in chromosome structure during *Begonia* evolution. Although karyomorphological data in *Begonia* are limited, many secondary constriction (SC) chromosomes have been detected. A SC chromosome consists of a satellite connected to the main body by the secondary constriction, a thin strip of the chromosome (Fig. 3). Oginuma & Peng (2002) showed that up to 63% of the chromosomes in a cell can be secondary or tertiary constriction chromosomes. Moreover, all 14 species of Taiwanese *Begonia* (including *B. palmata* and *B. aptera* with 2n = 22) surveyed possessed SC, except for *B. fenicis* (2n = 26), where the exact position of centromeres could not be determined for some chromosomes. Dewitte et al. (2009a) observed SC chromosomes in 6 out of 11 genotypes investigated. During prophase, the satellites were so loosely associated to their main body that they could easily be misidentified as separate chromosomes. Additional evidence for SC chromosomes

These data suggest that after polyploidisation, chromosome translocations occur, which are followed by a decrease in chromosome number and genome stabilisation (Oginuma & Peng,

Flow cytometric measurements of genome size, combined with chromosome counts, revealed a 12-fold difference in the mean chromosome size between the species *B. dietrichiana* and *B. pearcei* (Dewitte et al., 2009a). Moreover, as only 15 of the 66 sections of the genus *Begonia* were involved in this study it is likely that greater differences will be found when other sections are surveyed. Differences in chromosome size have also been

reported (Tobe & Raven, 1987).

**5.2 Chromosome structure** 

2002).

**5.3 Chromosome size** 

supports a basal number of x = 11 in Taiwanese *Begonia*.

originated out of a cross between 2n = 16 and 2n = 28.

in *Begonia* was presented by Legro & Doorenbos (1969).

observed in horticultural hybrids, including Elatior hybrids that are characterized by small *B. socotrana* and large *B. x tuberhybrida* chromosomes.

Apart from *B. pearcei* and *B. boliviensis*, South American species possess smaller chromosomes than Asian, African and Middle American species (Dewitte et al., 2009a). Different mechanisms, such as transposon activity, multiple deletions or other genetic rearrangements may have played an important role in the generation of this chromosome size variation (Devos et al., 2002; Kubis et al., 1998; Sanmiguel & Bennetzen, 1998). Bennetzen (2002) showed that more than 60% of some plant genomes consist of transposable elements and their defunct remnants.

Fig. 3. Prometaphase mitotic chromosome spread of *B*. 'Tamo' (2n = 23), stained with DAPI. Intense fluorescence signal indicates satellites (Dewitte et al., 2009a). Bar = 5 µm.
