Species Delimitation Of Asteropyrum Drumm. Et Hutch.(Rannuculaceae) Based On Morphological,Molecular And Ecological Variation

Species are the basic unit of biodiversity.Delimiting species boundaries correctly is crucial to the discovery of life's diversity because it determines whether or not different individual organisms are members of the same entity.According to Flora of China,there are two species under Asteropyrum Drumm.et Hutch,which are Asteropyrum peltatum Drumm.et Hutch and Asteropyrum cavaleriei Drumm.et Hutch.Based on our extensive field investigation,we found that the morphological variation between the two species was continuous and there is no obvious species boundary.This study attempted to adopt an integrative species concept and determine the boundary of the two species within Asteropyrum based on the evidence from DNA sequence variation,leaf morphological variation and ecological niche data.The results of this study are summarized as follows:1)We've collected a total of 27 populations of Asteropyrum plant specimens,with 4 to 6 plant specimens from each populations,and a total of 207 Asteropyrum specimens were collected.Based on the data of leaf shape variation,the morphological analysis was carried out by using geometric morphometrics.According to the results of morphological clustering,there is an obvious transition between Asteropyrum peltatum and Asteropyrum cavaleriei.2)We've collected a total of 28 populations within the distribution range of Asteropyrum in China,and extracted the total DNA of all samples.Two pairs of universal primers,ITS2-ITS5 and psbA-trnH,were selected.Specific primers were designed and synthesized according to transcriptome sequencing results,and a total of 10 pairs of nuclear gene primers were screened.Based on ITS2-ITS5 and psbA-trnH,we performed haplotype analysis,haplotype geographical distribution analysis and ABGD analysis respectively.Genetic structure analysis were performed based on 10 nuclear loci.The results were consistent and could not distinguish the two species from each other.3)We've collected the geographical distribution data of Asteropyrum,including the field sampling information of our laboratory and the distribution records of the species from the Chinese Virtual Herbarium.Sisty-six and 84 distribution points of Asteropyrum peltatum and Asteropyrum cavaleriei were obtained respectively.The 19 ecoclimatic factors of the modern era(1950-2000)are downloaded from the global climate database.Based on the climatic data of the two species,a niche modelling study and PCA analysis of bioclimatic variables was carried out,and the results showed that the niches of the two species largely overlapped.In summary,morphological,molecular systematic,population genetic and ecological evidences all show that the two species are not two independent species.The two species defined by the depth of leaf sinus and leaf size are two extreme representives of morphological variation,with obvious transition between species.The results of this study show that objectively evaluating several lines of evidence under the integrative species concept is the most efficient and theoretically grounded approach to defining robust species hypotheses.