**6. Hybridisation**

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

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;

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

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

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,

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

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

derived from two species with X=6 and 7 as their basic chromosome numbers.

shuffling, a major process for generating new genes (Long, 2001; Long et al., 2003).

Horn, 2004).

(Barba-Gonzalez et al., 2004; Dewitte et al., 2010b).

**5.5 Genome size variation** 

chromosomes is equal.

**5.6 Meiosis** 

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 maintaining the distinct identities of *Begonia* species.

The Origin of Diversity in *Begonia*:

Genome Dynamism, Population Processes and Phylogenetic Patterns 41

*B. listada* America *Pritzelia* 0.31 56 *B. echinosepala* var. *elongatifolia* America *Pritzelia* 0.32 56 *B. coccinea* America *Pritzelia* 0.56 56 *B. corallina* America *Gaerdtia* 0.78 56 *B. albo-picta* America *Gaerdtia* 0.58 56 *B. solananthera* America *Solananthera* 0.57 56 *B. luxurians* var. *ziesenhenne* America *Scheidweileria* 0.32 56 *B. odorata* America *Begonia* 0.56 52 *B. subvillosa* var. *leptotricha* America *Begonia* 0.29 34 *B. cucullata* America *Begonia* 0.30 34 *B. schmidtiana* America *Begonia* 0.38 34 *B. venosa* America *Begonia* 0.25 30 *B. ulmifolia* America *Donaldia* 0.25 30 *B. heracleifolia* America *Gireoudia* 0.75 28 *B. boliviensis* America *Barya* 1.46 28 *B. pearcei* America *Eupetalum* 1.46 26 *B. dregei* Africa *Augustia* 0.66 26 *B. grandis* var. *evansiana* Asia *Diploclinium* 0.68 26 *B. diadema* Asia *Platycentrum* 0.58 22

Table 1. Origin, classification, genome size and chromosome numbers of *Begonia* species

These results suggest post-pollination barriers to hybrid formation in *Begonia* are complex, as no strong trend can be seen between parental genome size, chromosome number and the area of origin in the likelihood that F1 hybrids can be obtained. This is in agreement with the

To assess the fertility of the hybrids, 5 to 10 of the F1 hybrid progeny were scored for pollen morphology and germination according to Dewitte et al. (2009b). The majority of hybrids examined were male sterile and either dropped their male flowers before opening or produced inviable pollengrains. Only a few combinations, mainly those between *B. albopicta*, *B. corallina* and *B. coccinea*, were male fertile (Table 3). They are all South American species with 2n = 56. *B. albo-picta* and *B. corallina* belong to the section *Gaerdtia* while *B. coccinea* belongs to the section *Pritzelia*. However, *B. coccinea* is morphologically very similar

In the second experiment, 12 species from the Central American section *Gireoudia* were crossed over a four year period (2005-2009) to investigate the cross compatibility of more closely related species. Of the 144 potential combinations between the parents, 92 (64%) have been attempted – the other combinations did not overlap in their flowering time. Of the 92 combinations attempted, 89 (97%) produced plump seeds, with little or no variability in success between repeats of the same cross. Only a single cross has failed to produce F1

used in the first experiment (classification from Dewitte et al., 2009).

many horticultural interspecific crosses described (Tebbitt, 2005).

to *B. corallina* and their sectional affinities may require reappraisal.

origin section Genome size

1C (pg DNA)

Chromosome number (2n)

Natural hybrids of recent origin can often be recognised by low pollen fertility, however fertile *Begonia* hybrids have also been observed (*B. decora* x *B. venusta*, Kiew et al., 2003; Teo & Kiew, 1999). The fertility of *Begonia* hybrids varies, and low hybrid fertility is likely to be due to genome divergence and incompatibilities between the progenitors. However, if F1 hybrids retain a low level of fertility then there is an opportunity for backcross progeny to be formed. Even if natural hybrids can be observed, further evidence is required to support the establishment of new hybrid species over time. For hybrid species to become established, they must become reproductively isolated from the parental species. Recent hybrids are likely to be swamped by well adapted parental species, however novel genetic combinations and the resulting heterosis (hybrid vigour) may explain their establishment (Lippman & Zamir, 2007; Paun et al., 2009)

Allopolyploid speciation, where hybridisation is combined with polyploidisation, offers a likely path to speciation, because the hybrid will have a high degree of post-zygotic reproductive isolation from their progenitors (Paun et al., 2009), due to the low fertility of triploid plants. Polyploid plants have a number of notable changes relative to their progenitors, including increased cell size, gene dosage effects, increased allelic diversity (level of heterozygosity), gene silencing and genetic or epigenetic interactions (Leitch & Bennett, 1997; Levin, 1983; Lewis, 1980; Osborn et al., 2003). The occurrence of viable unreduced gametes in *Begonia* hybrids has been described earlier, which is an important precursor for allopolyploid formation. Once polyploids are formed and established, they may enter an evolutionary trajectory of diploidization, a gradual conversion to diploidy through genetic changes that differentiate duplicated loci (Comai, 2005; Levy & Feldman, 2002).

To understand the potential role of hybridisation in *Begonia* evolution, we performed a series of cross-fertilization experiments. Firstly, the effects of parental ploidy level, genome size and geographic origin on seedling viability and fertility was investigated. This experiment allowed us to compare the effects of hybridizing species with highly differentiated genomes, and to understand the role of polyploidy and genome size changes in reproductive isolation in *Begonia*. Secondly, we investigated the cross compatibility of more closely related species (from the Central American Section *Gireoudia*), in order to assess the likelihood that hybridisation may occur between species with less differentiated genomes that are more likely to co-occur in the wild.

In the first experiment, we cross hybridised 19 mainly South-American *Begonia* species (Table 1). From the 156 cross combinations performed, pollinating 5 flowers per cross, 27 combinations generated viable hybrids (Table 2). Successful crosses were observed between species with different chromosome numbers and genome sizes (e.g. *B. cucullata* 2n = 34, 1C = 30 x *B. odorata* 2n = 52, 1C = 0.56 pg), and even between species from different continents (e.g. *B. dregei* x *B. coccinea; B. pearcei* x *B. grandis*).

The likelihood that F1 hybrids were obtained increased when the species used for crossing came from the same section (*Gaerdtia*, *Begonia*), although this was not the case for the section *Pritzelia*. For example, within the section *Begonia*, *B. cucullata*, *B. subvillosa* var. *leptotricha* and *B. schmidtiana* had equal chromosome numbers (2n = 34), while *B. odorata* contained a higher chromosome number (2n = 52). The genome sizes of *B. cucullata* and *B. subvillosa* var. *leptotricha* were similar (about 0.30 pg), but lower compared to those of *B. schmidtiana* (0.38 pg) and *B. odorata* (0.56 pg). Another species from the section *Begonia*, *B. venosa* (2n = 30; 1C = 0.25 pg), did not cross with these species.

Natural hybrids of recent origin can often be recognised by low pollen fertility, however fertile *Begonia* hybrids have also been observed (*B. decora* x *B. venusta*, Kiew et al., 2003; Teo & Kiew, 1999). The fertility of *Begonia* hybrids varies, and low hybrid fertility is likely to be due to genome divergence and incompatibilities between the progenitors. However, if F1 hybrids retain a low level of fertility then there is an opportunity for backcross progeny to be formed. Even if natural hybrids can be observed, further evidence is required to support the establishment of new hybrid species over time. For hybrid species to become established, they must become reproductively isolated from the parental species. Recent hybrids are likely to be swamped by well adapted parental species, however novel genetic combinations and the resulting heterosis (hybrid vigour) may explain their establishment

Allopolyploid speciation, where hybridisation is combined with polyploidisation, offers a likely path to speciation, because the hybrid will have a high degree of post-zygotic reproductive isolation from their progenitors (Paun et al., 2009), due to the low fertility of triploid plants. Polyploid plants have a number of notable changes relative to their progenitors, including increased cell size, gene dosage effects, increased allelic diversity (level of heterozygosity), gene silencing and genetic or epigenetic interactions (Leitch & Bennett, 1997; Levin, 1983; Lewis, 1980; Osborn et al., 2003). The occurrence of viable unreduced gametes in *Begonia* hybrids has been described earlier, which is an important precursor for allopolyploid formation. Once polyploids are formed and established, they may enter an evolutionary trajectory of diploidization, a gradual conversion to diploidy through genetic changes that differentiate duplicated loci (Comai, 2005; Levy & Feldman,

To understand the potential role of hybridisation in *Begonia* evolution, we performed a series of cross-fertilization experiments. Firstly, the effects of parental ploidy level, genome size and geographic origin on seedling viability and fertility was investigated. This experiment allowed us to compare the effects of hybridizing species with highly differentiated genomes, and to understand the role of polyploidy and genome size changes in reproductive isolation in *Begonia*. Secondly, we investigated the cross compatibility of more closely related species (from the Central American Section *Gireoudia*), in order to assess the likelihood that hybridisation may occur between species with less differentiated

In the first experiment, we cross hybridised 19 mainly South-American *Begonia* species (Table 1). From the 156 cross combinations performed, pollinating 5 flowers per cross, 27 combinations generated viable hybrids (Table 2). Successful crosses were observed between species with different chromosome numbers and genome sizes (e.g. *B. cucullata* 2n = 34, 1C = 30 x *B. odorata* 2n = 52, 1C = 0.56 pg), and even between species from different continents

The likelihood that F1 hybrids were obtained increased when the species used for crossing came from the same section (*Gaerdtia*, *Begonia*), although this was not the case for the section *Pritzelia*. For example, within the section *Begonia*, *B. cucullata*, *B. subvillosa* var. *leptotricha* and *B. schmidtiana* had equal chromosome numbers (2n = 34), while *B. odorata* contained a higher chromosome number (2n = 52). The genome sizes of *B. cucullata* and *B. subvillosa* var. *leptotricha* were similar (about 0.30 pg), but lower compared to those of *B. schmidtiana* (0.38 pg) and *B. odorata* (0.56 pg). Another species from the section *Begonia*, *B. venosa* (2n = 30; 1C

(Lippman & Zamir, 2007; Paun et al., 2009)

genomes that are more likely to co-occur in the wild.

(e.g. *B. dregei* x *B. coccinea; B. pearcei* x *B. grandis*).

= 0.25 pg), did not cross with these species.

2002).


Table 1. Origin, classification, genome size and chromosome numbers of *Begonia* species used in the first experiment (classification from Dewitte et al., 2009).

These results suggest post-pollination barriers to hybrid formation in *Begonia* are complex, as no strong trend can be seen between parental genome size, chromosome number and the area of origin in the likelihood that F1 hybrids can be obtained. This is in agreement with the many horticultural interspecific crosses described (Tebbitt, 2005).

To assess the fertility of the hybrids, 5 to 10 of the F1 hybrid progeny were scored for pollen morphology and germination according to Dewitte et al. (2009b). The majority of hybrids examined were male sterile and either dropped their male flowers before opening or produced inviable pollengrains. Only a few combinations, mainly those between *B. albopicta*, *B. corallina* and *B. coccinea*, were male fertile (Table 3). They are all South American species with 2n = 56. *B. albo-picta* and *B. corallina* belong to the section *Gaerdtia* while *B. coccinea* belongs to the section *Pritzelia*. However, *B. coccinea* is morphologically very similar to *B. corallina* and their sectional affinities may require reappraisal.

In the second experiment, 12 species from the Central American section *Gireoudia* were crossed over a four year period (2005-2009) to investigate the cross compatibility of more closely related species. Of the 144 potential combinations between the parents, 92 (64%) have been attempted – the other combinations did not overlap in their flowering time. Of the 92 combinations attempted, 89 (97%) produced plump seeds, with little or no variability in success between repeats of the same cross. Only a single cross has failed to produce F1

The Origin of Diversity in *Begonia*:

73% of hybrids were pollen sterile.

unpublished data).

Genome Dynamism, Population Processes and Phylogenetic Patterns 43

percentage of well stained pollen scored. The mean pollen viability was 3.5% (±1.9%), and

cross % bad, shrunken

Table 3. Pollen fertility of fertile F1 hybrid combinations (means ± SD; n = 5). Only

Fig. 4. Interspecific crossing barriers in the section *Gireoudia*. Leaves on a dark background indicate a successful F1 cross. A red square indicates an unsuccessful cross and a yellow background a cross that has worked in one direction only (Twyford & Kidner, previously

combinations producing male fertile progeny are included.

*B. albo-picta B. corallina* 7,6 ± 2,9 48,4 ± 21,0 *B. albo-picta B. coccinea* 4,5 ± 3,7 39,6 ±28,5 *B. coccinea B. albo-picta* 8,0 ± 5,2 45,3 ± 20,0 *B. coccinea B. corallina* 15,0 ± 6,5 41,5 ± 17,6 *B. corallina B. coccinea* 13,0 ± 6,9 50,0 ± 36,7 *B. corallina B. albo-picta* 18,8 ± 6,5 38,5 ± 15,4 *B. echinosepala B. luxurians* 98,6 ± 0,6 0,4 ± 0,3

pollen grains % germination ♀ ♂

seeds in both directions (*B. theimei x B. heracleifolia)* and two crosses have failed in one direction only (*B. peltata x B. serioceneura, B. nelumbiifolia x B. heracleifolia*). However, these are based on a single parental accession and few replicates, and this requires further crosses to confirm this result. Overall, the ability to form F1 hybrids between related *Begonia* species with the same chromosome number (2n=28; the common chromosome number in this section), further supports weak postzygotic reproductive isolating barriers, as shown in Figure 4.


Table 2. Outcomes of interspecific crosses. Combinations resulting in viable hybrid are marked with 'o', combinations that did not result in seedlings with an 'x'.

We then germinated the seeds from each seed capsule, to test for evidence of seedling mortality and the fertility of the hybrids. No evidence of seedling mortality was observed, that would indicate incompatible genome interactions which affect fitness at early growth stages (hybrid dysgenesis). Successful crosses were grown to maturity, and the 19 F1 hybrids that flowered in the Spring 2009 were scored for pollen viability. For each accession 100 pollen grains were stained with fluorescein diacetate (FDA) in 5% sucrose solution, and

seeds in both directions (*B. theimei x B. heracleifolia)* and two crosses have failed in one direction only (*B. peltata x B. serioceneura, B. nelumbiifolia x B. heracleifolia*). However, these are based on a single parental accession and few replicates, and this requires further crosses to confirm this result. Overall, the ability to form F1 hybrids between related *Begonia* species with the same chromosome number (2n=28; the common chromosome number in this section), further supports weak postzygotic reproductive isolating barriers, as shown in

Figure 4.

♂

*B. listada* 

*B. venosa* **x** 

 *B. echinosepala* 

 *B. coccinea* 

 *B. corallina* 

 *B. albo-picta* 

*B. solananthera* **x o x x x x x** 

*B. subvillosa* **x x x x x o o x o** 

*B. ulmifolia* **x x x x x x x x x**  *B. heracleifolia* **x x x x x x x x x x x** 

*B. diadema* **x** 

marked with 'o', combinations that did not result in seedlings with an 'x'.

*B. schmidtiana* **o** 

*B. boliviensis* **o x x** 

*B. grandis* **x** 

 *B. solananthera* 

 *B. luxurians* 

 *B. odorata* 

*B. listada* **x x x x x x x x x x x x x x x x**  *B. echinosepala* **x x x x o x x x o x x x**  *B. coccinea* **x o o o x x x o x x x**  *B. corallina* **x o o o x x x x x x x x o x x**  *B. albo-picta* **x o o o x x x x x x x x** 

*B. luxurians* **x x x x x x x x x x x**  *B. odorata* **x x x x x x o o x x x o x x** 

*B. cucullata* **x x x x x o o x x x x** 

*B. pearcei* **x x o**  *B. dregei* **o o x x x x x x** 

Table 2. Outcomes of interspecific crosses. Combinations resulting in viable hybrid are

We then germinated the seeds from each seed capsule, to test for evidence of seedling mortality and the fertility of the hybrids. No evidence of seedling mortality was observed, that would indicate incompatible genome interactions which affect fitness at early growth stages (hybrid dysgenesis). Successful crosses were grown to maturity, and the 19 F1 hybrids that flowered in the Spring 2009 were scored for pollen viability. For each accession 100 pollen grains were stained with fluorescein diacetate (FDA) in 5% sucrose solution, and

 *B. subvillosa* 

 *B. cucullata* 

 *B. schmidtiana* 

 *B. venosa* 

 *B. ulmifolia* 

 *B. heracleifolia* 

 *B. boliviensis* 

 *B. pearcei* 

 *B. dregei* 

 *B. grandis* 

 *B. diadema* 

♀


percentage of well stained pollen scored. The mean pollen viability was 3.5% (±1.9%), and 73% of hybrids were pollen sterile.

Table 3. Pollen fertility of fertile F1 hybrid combinations (means ± SD; n = 5). Only combinations producing male fertile progeny are included.

Fig. 4. Interspecific crossing barriers in the section *Gireoudia*. Leaves on a dark background indicate a successful F1 cross. A red square indicates an unsuccessful cross and a yellow background a cross that has worked in one direction only (Twyford & Kidner, previously unpublished data).

The Origin of Diversity in *Begonia*:

QTLs.

studies.

diversity.

**8. References** 

Genome Dynamism, Population Processes and Phylogenetic Patterns 45

A second approach works in the other direction, from the trait down to the locus. Once a genetic map has been constructed, quantitative trait locus (QTL) analysis can reveal the genetic architecture of species level differences: the number of loci that affect a trait, along with their sizes and interactions (Zeng, 2005). The traits most divergent between species may be related to the selective forces that drove speciation and can be linked to plant fitness as measured by seed production or to relative growth rate (Taylor et al., 2009). An ideal experiment would combine both approaches, mapping highly divergent genes relative to

One of the great advantages of *Begonia* for this work is the parallel evolution that is common in the genus on many levels. Given sufficient genetic resources, comparisons can be made between the genetic architecture of traits independently evolved within and between sections and continents. This allows hypothesises about trait evolution to be tested using multiple replicates, providing greater robustness than is usually possible for evolutionary

One topic that has yet to be studied in depth and deserves further attention is natural hybridisation and hybrid speciation. Hybrid speciation seems very likely within the genus, and interspecific processes may be the cause of the hard incongruence of phylogenetic trees derived from plastid and nuclear ribosomal data within some species-rich sections (*Platycentrum*, *Petermannia*) (Goodall-Copestake (2010), Thomas (2010). However, homoploid hybrids are hard to detect; and only a handful of species in evolutionary model systems have proven to be hybrids without a change in ploidy level (e.g. *Helianthus, Argyranthemum, Ceanotus, Pinus* (listed in Gross & Rieseberg, 2005). Investigation of the frequency of natural hybridisation, together with studies of genome stabilisation after polyploidisation and more karyomorphological data may be useful to understand chromosome evolution within the genus. The use of molecular techniques, such as comparative transcriptional profiling or targeted genome resequencing of species and their hybrids (see Twyford & Ennos, 2011), or cytological techniques such as GISH, may shed light on the role of natural hybridisation. The genus *Begonia* has been studied for many reasons, including: horticulture, taxonomy, as an indicator for biogeographic variation, to understand the development of their distinctive leaf forms and their aberrations, population genetics of endemic species, hybridisation and genome dynamics. As the distinction between model plants with extensive genetic resources, and non-models without these resources becomes less well defined, we expect that *Begonia* will continue to provide insights into the nature and origin of tropical plant

Ågren, J. & Schemske, DW. (1993). Outcrossing rate and inbreeding depression in two

APG (2009). An update of the Angiosperm Phylogeny Group classification for the orders

Arends, JC. (1970). Somatic chromosome numbers in 'Elatior'-Begonias. *Mededelingen Landbouwhogeschool Wageningen*, Vol.70, No.20, pp. 1-18, ISSN 0369-0598

(February 1993), pp. 125–135, ISSN 1558-5646

Vol.161, No.2, (October 2009), pp. 105-121, ISSN 0024-4074

annual monoecious herbs, *Begonia hirsuta* and *B. semiovata*. *Evolution*, Vol.47, No.1,

and families of flowering plants: APG III. *Botanical Journal of the Linnean Society*,

These results highlight that interspecific *Begonia* crosses have a significantly reduced pollen fertility. There are a number of notable exceptions, particularly in F1 crosses in the section *Gireoudia*, where pollen stainability is above 30% (Matthews, unpublished). This includes crosses between divergent species found in different geographical areas, such as *B. conchifolia* and *B. plebeja*. Hybrids with good pollen fertility were also observed in section *Gaerdtia* (see above). Overall, this suggests that hybrid fertility is constraining in the formation of later generation hybrids in the wild. However, male fertility is not necessarily linked to female fertility as different genes may underlie male and female meiosis. Some of the male sterile hybrids have been successfully used as a seed parent in crosses (Dewitte et al., 2010a; Twyford & Kidner unpublished data).

The role of hybrid speciation in *Begonia* should be investigated further, but several preconditions for hybrid speciation are fulfilled in this genus: weak crossing barriers, a very labile genome, the presence of natural interspecific hybrids and the occurrence of 2n gametes. The fertility of artificial hybrids is very variable and depends on the cross combination. If a hybrid retains some fertility and becomes isolated from its parent plant, hybrid speciation may be a possible outcome.
