Date Published: January 30, 2017
Publisher: Public Library of Science
Author(s): Ying Han, Yuan Liu, Haoyou Wang, Xiangjun Liu, Tzen-Yuh Chiang.
Vicia ramuliflora L. is a widely distributed species in Eurasia with high economic value. For past 200 years, it has evolved a tetraploid cytotype and new subspecies at the diploid level. Based on taxonomy, cytogeography and other lines of evidence, previous studies have provided valuable information about the evolution of V. ramuliflora ploidy level, but due to the limited resolution of traditional methods, important questions remain. In this study, fluorescence in situ hybridization (FISH) and random amplified polymorphic DNA (RAPD) were used to analyze the evolution of V. ramuliflora at the diploid and tetraploid levels. Our aim was to reveal the genomic constitution and parents of the tetraploid V. ramuliflora and the relationships among diploid V. ramuliflora populations. Our study showed that the tetraploid cytotype of V. ramuliflora at Changbai Mountains (M) has identical 18S and 5S rDNA distribution patterns with the diploid Hengdaohezi population (B) and the diploid Dailing population (H). However, UPGMA clustering, Neighbor-Joining clustering and principal coordinates analysis based on RAPD showed that the tetraploid cytotype (M) has more close relationships with Qianshan diploid population T. Based on our results and the fact that interspecific hybridization among Vicia species is very difficult, we think that the tetraploid V. ramuliflora is an autotetraploid and its genomic origin still needs further study. In addition, our study also found that Qianshan diploid population (T) had evolved distinct new traits compared with other diploid populations, which hints that V. ramuliflora evolved further at diploid level. We suggest that diploid population T be re-classified as a new subspecies.
Vicia ramuliflora (Maxim.) Ohwi belongs to the family Fabaceae, tribe Fabeae Rchb. ex Kitt., syn. Vicieae Bronn., genus Vicia L. and sect. Vicilla (Schur) Aschers. et Graebn. ex Kupicha. It is a perennial herbaceous species that is widely distributed in Eurasia, especially in East Asia, including Japan, Siberia, Mongolia, China, and Korea. The species of the genus Vicia are considered important economically, because they provide energy and protein for livestock [1,2]. They can either be grazed as fresh forage  or can be cut and preserved as hay or silage [1,2]. Moreover, they also can be used for green manure . Many Vicia species including V. ramuliflora also are important as herbaceous medicinals . Cytotaxonomic studies [6,7,8] showed that V. ramuliflora and its closely related Vicia species only included diploid (2n = 2x = 12) cytotype in Japan, Russia and Korean Peninsula, but evolved diploid (2n = 2x = 12) and tetraploid (2n = 2x = 24) cytotypes in China. The tetraploid cytotypes are discontinuously distributed in the Changbai Mountains (Northeast China) and Huangshan Mountains, Tianmu Mountains and Lu Mountains (Easten China). In Northeast China, the diploid form of V. ramuliflora is widespread. However, its conspecific tetraploid counterpart is only endemic to the margins of forests and subalpine meadows of Changbai Mountains at altitudes above 1500–2000 m. Previous fieldwork  indicated that the population of the tetraploid form was well-developed, consisting of thousands of individuals. However, when tetraploid individuals of V. ramuliflora were transplanted to lower altitude regions, their sexual reproduction became abnormal (i.e. the flower buds fall earlier than usual and therefore do not set fruit). Li et al.  found that diploid and tetraploid V. ramuliflora populations showed similar karyotypes and had no distinct cytological genetic markers. Hence, the genome components, evolutionary relationships and likely diploid parents of these tetraploid populations could not be drawn out through classical karyotype data and conventional crossing experiments.
Our FISH-based study showed that the tetraploid cytotype of V. ramuliflora at Changbai Mountains (M) had identical 18S and 5S rDNA distribution pattern with the diploid Hengdaohezi population (B) and the diploid Dailing population (H). However, UPGMA clustering, Neighbor-Joining clustering, principal coordinates analysis based on RAPD showed that the tetraploid cytotype (M) had close relationships with diploid population T. Based on our study results and the fact that interspecific hybridization among Vicia species is known to be limited, we think that the tetraploid V. ramuliflora is autotetraploid, but its likely parents still need to be studied further. In addition, our study also found that Qianshan diploid population (T) had evolved distinct 5s rDNA sites compared with other diploid populations. Specific band pattern, genetic distances, UPGMA clustering, principal coordinates analysis based on RAPD also separated Qianshan diploid population (T) from other diploid populations (B and H). We, therefore, suggest that diploid population T be re-classified as a distinct subspecies.