Investigation On The Taxonomic Status Of Viola Bulbosa And V.tuberifera (Violaceae)

Due to geographical distribution and morphology of V.bulbosa and V.tuberifera is similar,and the breeding system is similar,but leaf morphology of V.bulbosa and V.tuberifera has significant differences,The taxonomical position of V.bulbosa and V.tuberifera endemic to the Qinghai-Tibet Plateau is still controversial.The morphological variation of leaves from the digital specimen of V.bulbosa and V.tuberifera were analyzed,the sympatric natural polulation of V.bulbosa and V.tuberifera located in Qinghai-Tibetan Plateau were analyzed using morphological study,ITS and psb A-trn H sequence analysis and SSR molecular marker.The taxonomical position was analyzed using morphological variation and genetic variation.The results are as follows:1.The PCA analysis based on leaf morphology of digital specimen indicated that all specimens is divided into two parts,represented for V.tuberifera and V.bulbosa respectively.The variation of leaf morphology within populations or among populations showed that that some parameters ?W,AL,BL and LWR,which is reflect the morphology of leaf apex and base,but the difference between V.bulbosa and V.tuberifera is significant(P<0.01).The PCA analysis of sympatric natural populations showed the same results.Therefore,V.tuberifera and V.bulbosa can be classified clearly based on the leave shape and should be treated as different taxon.V.bulbosa: leaves oblong ovate,apex acute,base cuneate or shallowly cordate;V.tuberifera: leaves reniform,apex obtuse,base cordate.2.The ITS sequences and psb A-trn H sequences of 11 individuals in different regions of V.bulbosa and V.tuberifera was sequenced,then the phylogenetic tree based on two sequences were constructed by Neighbor-joining(NJ)methods,respectively.The results showed that the V.bulbosa and V.tuberifera clustered into two branches,and belong to the same distribution of V.bulbosa and V.tuberifera had a distant genetic relationship.The Violaceae phylogenetic tree based on ITS sequences and psb A-trn H sequences were constructed by Neighbor-joining(NJ)methods,respectively.The results showed that the V.bulbosa and V.tuberifera wereclustered in the same branch based on a closer relationship,but the genetic distance level is still above species.And V.bulbosa and V.tuberifera were clustered with other plants of Sect.Adnatae in different branches,although the V.bulbosa and V.tuberifera belong to the Sect.Adnatae in the classification of the original,actually the V.bulbosa and V.tuberifera should be removed from the Sect.Adnatae.3.Population genetic diversity of the 6 populations was analyzed using SSR molecular marker.The results indicated that the population genetic diversity of V.tuberifera are higher than V.bulbosa,and the genetic differentiation among population of V.tuberifera are lower than V.bulbosa,UPGMA clustering showed that the 6 populations were clustered into branches,and the PCo A analysis result is similar,it's indicated the boundaries between V.bulbosa and V.tuberifera is obvious.Based on the above studies,the boundaries between V.bulbosa and V.tuberifera are obvious in leaf morphology,genetic diversity and genetic structure,The phylogenetic tree based on ITS sequences and psb A-trn H sequences showed that V.bulbosa and V.tuberifera have different evolutionary directions.Therefore,V.bulbosa and V.tuberifera should be treated as two species in future research.