Hybrid origin of Kushui Rose
Homoploidyhybridization seems to play an important role to form new species by reassembling genomes without changes in ploidy number in the genus of Rosa, such as R. pseudobanksiaeandpart of edible roses (Zhang et al. 2020; Cui et al. 2022). According toChinese distinguished plant taxonomists Junde Yu and Cuizhi Ku’s observation, Kushui Rose was proposed as the product of natural hybridization betweenR. rugosa and R. sertatais, and it was scientifically named asR. Sertate × R. RugosaYu. et. Ku(Ma et al. 1985).Unfortunately,the two scientistshadn’t published any available data about the topic so left it still an open question.If Yu & Ku’s proposal is true, Kushui might originate from a homoploidization event, becauseKushui Rose (Zhao et al. 2007), R. rugosaand R. sertatais (Fang 2020) were all identified as diploid in karyotype analysis, with the number of chromosomes 2n=14.
In fact, at Yu & Ku’s time, only depending on morphological traits to identify a hybrid species without ploidy is a very difficult task (Rieseberg 1997;Rieseberg et al. 1990). Generally regarding, there are some intermediate morphological characters between hybrid species and their parents. Kushui Rose exhibited a lot of morphological characters that are of the intermediate to R. rugosa and R. sertata, such as the number of leaflets and petal count, Fig. 5. However, the characters in Kushui Rose could be also observed intermediate to R. rugosa and R. willmottiae, such as the number of leaflets, petal count, coverage of flowers and stipule shape. Its twig color, petal apex and calyx tube shape were consistent with R. willmottiae and R. rugosa ‘YanXia’, Fig. 5. So only based on the morphological characters there is no clear answer for the question whether Kushui Rose was a hybrid species between R. rugosa and R. sertatais, or between R. rugosa and R. willmottiae. On the other hand, Male sterility due to meiosis failure to form vigorous microspores was recently observed in Kushui Rose, quite consistent with its assumed identity of hybrid species (Wang et al. in press).
Species with hybrid origin usually exhibit additivity of parental nuclear genomes, therefore the additivity of nuclear gene sequences alignment and the incongruency in further phylogenetic analysis are the most important evidences for identifying homoploidy hybridization (Sun 2003; Zhang 2005). The present nuclear sequences alignment results indicate that ITS and GAPDH sequences of Kushui Rose showed perfect additivity at 5 and 7 sites between R. rugosa‘YanXia’ and R. willmottiae (TZ, while Kushui Rose exhibited additivity at only 4 sites between R. rugosa‘YanXia’ and R. sertata. These results indicate strongly that Kushui Rose as arisen through hybridization of R. rugosa‘YanXia’ and R. willmottiae (TZ). In nuclear trees, two haplotypes from Kushui Rose were closely related with R. rugosa ‘YanXia’ and R. willmottiae (TZ) respectively (Fig. 2 and Fig. 3), while R.sertata did not formed a monophyletic clade with Kushui Rose, suggesting that Kushui Rose origins from two different parents,R. rugosa ‘YanXia’ and R. willmottiae (TZ). In addition, only R. willmottiae (TZ) with Kushui Rose clustered an inner clade in phylogenetic analyses ofcpDNAsequences, and neither R. rugosa ‘YanXia’ nor R.sertata clustered closely to Kushui Rose, in which R.sertata formed a single branch. The significant differences between nuclear and chloroplast trees are considered to be strong evidence for hybridization events (Costea et al. 2010; Gruenstaeudl et al. 2012). Although the inconsistency of nuclear gene tree and chloroplast gene trees might also be derived from the other historical events such as lineage sorting (Rieseberg 1997), in this study, multiple unlinked loci producing phylogenetic trees with the similar topological feature could give a relatively solid conclusion of homoploid hybridization to some extent.
In sum, the morphological analysis in this study together with the previous study of male sterility in cytological level support Yu & Ku’s proposal of Kushui Rose as a hybrid species. On the other hand, our further molecular biological analysis, including additivity among nuclear DNA sequences alignment as well as their incongruent positions in the same phylogeny, and the incongruency of nuclear gene tree and chloroplast gene trees, evidenced that Kushui Rose was the product of natural homoploid hybridization between R. rugosa and R. willmottiae, rather than between R. rugosaand R. sertata. Thus, its scientific name should be R. willmottiae× R. rugosa.
Parental analysis of hybrid Kushui Rose
Chloroplast genome is usually maternally inherited and is not affected by hybridization factors in angiosperm, which is often used to identify the maternal origin of the hybrid.In phylogenetic analyses of the cpDNA sequence, R. willmottiae (TZ) was clustered with Kushui Rose, therefore we further believed that R. willmottiae (TZ) and R. rugosa ‘YanXia’ should be the female parent and male parent, respectively.
In terms of distribution area, Kushui Rose distributes in Kushui, Yongdeng, Gansu. R. willmottiae distributes in Gansu, Sichuan, Shaanxi and Qinghai, which grows in shrub, hillside or roadside at an altitude of 1300-3150 meters, and flowering period is May-June.R. rugosa distributes throughout China, and flowering period is May-June (Flora of China Editorial Committee of Chinese Academy of Sciences 1985). The habitat and distribution area of R. willmottiae overlap with R. rugosa, and the flowering period is the same. In addition,we also found R. Willmottiae and R. Rugosa in the area near Kushui town. Accordingly, we speculated that R. willmottiae and R. rugosa have frequent gene exchanges in Kushui town or upstream areas, and eventually form a natural hybrid - Kushui Rose.
Wang (in press) proved that Kushui Rose and R. rugosa have cross-compatibility through hybridization experiments, but there has been no report on cross hybridization between R. rugosa and R. willmottiae. Kushui Rose has cross-compatibility with R. rugosa, which means that it has not formed reproductive isolation between paternal parent. Other studies have found that Kushui Rose is unable to bear fruit by self-crossing, let alone obtain fertile seeds (Ma et al. 1985; Wang et al. inpress). Where the two species meet, these hybrids may form a stable hybridization zone that serves as an effective barrier with the parent species. At the same time, backcrossing with parent or hybrid dysgenesis may prevent the process of homoploid hybrid species formation. In general, only after a group overcomes seed sterility and produces reproduction and niche isolation with its parents can the offspring of these hybrids form a new self-evolving lineage (Zhang et al. 2020). Combined with the analysis of this study, it is shown that Kushui Rose has not formed an obvious genetic pedigree.