# The Chromosome Number and rDNA Loci Evolution in _Onobrychis_ (Fabaceae)
## 3 Discussion  
The analyses of chromosome number and rDNA loci chromosomal organisation in the phylogenetic framework enable insight into the trends in chromosomal evolution that accompany or follow diversification and speciation in plants, especially in wild or non-model taxa such as _Chenopodium_, _Crepis_, _Prospero_ and _Allium_[18, 35, 36]. Although recently, _O. vicifolia_ has started to attract more attention as a fodder crop, relatively little research has been conducted on _Onobrychis_ genome structure and evolution. In earlier reports, two basic chromosome numbers, \(x=7\) and \(x=8\), were observed among analysed representatives of this genus [6, 7]. The same basic chromosome numbers were also reported for the closely related genus _Hedysarum_[37]. More than one basic chromosome number was reported in several other genera from the Fabaceae family (e.g., _Vicia_, _Phaseolus_ and _Lotus_) [38, 39, 40] as well as in other plant families, e.g., in _Crepis_ from Asteraceae or _Passiflora_ from Passifolia-raceae [16, 41]. Relatively many reports were published regarding chromosome numbers in _Onobrychis_. However, the chromosome counts for several species differed among publications, e.g., for the same species, both diploid and tetraploid chromosome numbers were reported (e.g., _O. crista-galli_ and _O. caput-galli_) [7, 42, 43] (Table S1 [6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56]). Different basic chromosome numbers were reported for some _Onobrychis_ species, e.g., \(x=7\) and \(x=8\) for _O. ptolemaa_[7, 10] (Table S1). Intrspecific polymorphisms in chromosome number concerning ploidy levels were published in several different genera, e.g., _Prospero autumnale_, _Deschampsia cespiosa sensu lato_ and _Crepis veisaria_[57, 58, 59]. Few reports also showed that some species consist of cytotypes which differ in basic chromosome number, which could be caused by aneuploidy, e.g., trisomy of one chromosome pair in _Amananthus caudatus_[59] and dysploidy in, e.g., _Prospero autumnale_ complex [36]. However, technical issues could also cause incongruence between different reports. _Onobrychis_ species have relatively small chromosomes, and most of the chromosome counts were based on simple techniques like Feulgen or acetoorcein/acetocarmine staining (e.g., [10, 37]). In karyotypes with small and numerous chromosomes, these methods could not allow precise identification of specific chromosomes and assigning them into homologous pairs. In the case of _Onobrychis_, the differences between chromosome reports could also be attributed to the highly complex taxonomy of this genus. Different phylogenetic approaches to species delimitation resulted in varying numbers of recognised species [1]. Thus, all the analyses in our study were carried out on the same individuals of the species, enabling optimal correlation of the molecular and cytogenetic data. Phylogeny based on the biparentally inherited nrITS of analysed _Onobrychis_ species was largely congruent with previously published results [5, 60]. In the present study, all analysed species from subgenus _Onobrychis_ were included in one subclade, as earlier shown, based on nrITS and plastid markers [5, 60, 61]. The second subgenus _Sisyrosema_, previously reported as monophyletic [5, 60], was recovered in two separate clades in the present study. This incongruence might be due to technical reasons because each analysis was based on different sets of species. In every report published up to date, the analysed species accounted for 20% to 40% of all species included in this genus [5, 60, 61]. Moreover, some nodes in nrITS phylogram in subgenus _Sisyrosema_ were weakly supported. The two accessions of tetraploid _O. vicifolia_ from subgenus _Onobrychis_ were included in two different evolutionary lineages. One of these accessions was a cultivated form, whereas the other was a wild plant.