# The Chromosome Number and rDNA Loci Evolution in _Onobrychis_ (Fabaceae)
## 2 Results
### Evolutionary Patterns of the rDNA Loci Chromosomal Organisation  
The number and localisation of rDNA loci were analysed using FISH for diploids from clade I. The obtained data were mapped on the ML phylogenetic tree using the maximum likelihood reconstruction methods. The analyses resulted in the reconstruction of one locus of 35S rDNA in the interstitial position in the chromosome and one locus of 5S rDNA as an ancestral state of clade I and both distinguished subclades Ia and Ib (Figure 4). Unlike for the 35S rDNA locus, the ancestral state of chromosomal localisation of the 5S rDNA loci was ambiguously retrieved. The ancestral state of 35S rDNA locus number and localisation was observed in most species in the Ib clade. Gain of the 35S rDNA locus accompanied speciation of only one species, _O. vaginalis_ (Figure 4A). In subclade Ib, all species also showed an ancestral state, one locus of 5S rDNA. In karyotypes of most species from this subclade, this locus was in the subterminal position (Figure 4C). The analyses suggested one or two repositioning events of 5S rDNA loci during the evolution of the subclade Ib (_O. hvapgray_ and _O. grandis_; Figure 4B). One interstitial locus of 35S rDNA and one locus of 5S rDNA were also reconstructed as an ancestral state for subclade Ia. However, the patterns of rDNA loci evolution were more complicated in this subclade. The ancestral state, one locus of 35S rDNA was observed in four out of eight species (_O. iberica_, _O. supina_, _O. caput-gali_ and _O. gracilis_; Figure 4C). The evolution of the remaining species was accompanied by a gain of one locus (_O. persica_, _O. alba_ subsp. _laconica_ and _O. megataphorus_) or two loci (_O. crista-galli_) of 35S rDNA (Figure 4A). The diversification of these species was also accompanied by at least two events of 35S rDNA loci repositioning  
Figure 3: Ancestral character state reconstruction of the basic chromosome numbers of the analysed species of _Onobrychis_. The chromosome numbers have been mapped on the ML tree of nrITS sequences using the maximum likelihood method implemented in ChromEvol 2.0 software. The tree was rooted with _Hedysarum candidissimum_.  
from the interstitial (ancestral state) to the subterminal localisation (Figure 4B). The ancestral number, one locus of 5S rDNA, was present only in the karyotype of two species, _O.__iberica_ and _O.__caput-galli_ from subclade Ia. The gain of 5S rDNA loci was retrieved for the common ancestor of most diploids from subclade Ia (except _O.__iberica_, which has the ancestral number of loci), followed by the loss of 5S rDNA loci during speciation of _O.__caput-galli_ (Figure 4C). Additionally, repositioning of 5S rDNA locus may also be suggested to accompany the speciation of _O.__iberica_ (species with subterminal 5S rDNA locus) or the evolution of all other species from subclade Ia (at least one locus of 5S rDNA in interstitial localisation). Moreover, three diploids (_O.__megataphys_, _O.__crista-galli_ and _O.__alba_ subsp. _laconica_) that had duplicated (or triplicated) numbers of both rDNA loci included in their karyotypes a chromosome bearing both 3S5 and 5S rDNA loci (Figure 4).  
Figure 4: Ancestral character state reconstruction of the rDNA locus number and localisation for _Onobrychis_ species. The numbers and localisation of the rDNA loci were mapped onto the ML tree of the nrITS sequences using maximum likelihood methods. **(A)** The number of 35S rDNA loci. **(B)** Localisation of 35S rDNA loci. **(C)** The number of 5S rDNA loci. **(D)** Localisation of 5S rDNA loci. The tree was rooted with _Hedysarum candidissimum_, which was subsequently removed from the figure.