Delimitation And Identification Of Species Within Three Species Complexes Of Saussurea(Asteraceae)

Saussurea(Asteraceae)is one of the largest genera of Cardueae.About 500 species are currently recongized,mostly distributed in the highlands of the northern hemisphere.In China,this genus is among the top ten specious genera and about 350 species are recorded,including many famous medicinal plants.Moreover,some species might be unique and potentially valuable given the preference of extremely harish habitats.However,many species are difficult to identify or circumscribe due to highly similiar to multiple species in morphology,and seriously affect the investigation,identification,utilization and protection of the resources.In order to provide reasonable delimitation of these problematic species,the present study analyzed the morphological as well as genetic diversities of the members from three complexes using the data from nuclear ITS and five chloroplast DNA fragments.The results show that the inconsistency between morphological and genetic differentiation is common within three complexes and many species may be “on the way” of diversification following the rule of “the integrative species concept”.At the same time,several cases of hybridizations and radiative diversification were documented among some species.We proposed that these mechanisms may contribute to the main taxonomic controversies within these complexes,as listed below.1.S.scabrida complex.The leaf blades of the members of this complex,all from subg.Saussurea sect.Strictae,are runcinate-pinnately lobed.The diffenence among species are mainly the shape of leaves,lobes and phyllaries and the variation on trichomes.However,these traits are variable within the species,resulting in the difficult in identification of species and the blurred boundaries among species.We conducted field investigations on 9 species of the complex,and collected 71 individuals from 45 populations.Using DNA fragments of ITS,mat K,psb A-trn H and trn K,separately or in combination,we calculated the intraspecific and interspecific genetic distance and phylogenetic trees together with the representative species of the genus Saussurea.In combination with the data of morphology and geography,our main results and inferrences are as follows: 1)The nine species clustered into a monophyly on the nuclear ITS phylogenetic tree,indicating that the members of the complex are close to each other,in line with their morphological similarity;2)Three species,i.e.,S.bodinieri,S.scabrida and S.semilyrata,are recovered to be monophyletic in the nuclear gene phylogenetic tree,but the genetic differentiation is low,indicating that these three speces might dierived from recent radiation;3)Six speces,i.e.,S.eriostemon,S.pseudoingulata,S.subulisquama,S.pseudoeriostemo,S.lingulata and S.pachyneura,clustered into a clade in the phylogenetic trees based on both ITS and the combined chloroplast fragments,but the sampled individuals of each species do not cluster into any branch specific to species,inconsistent with morphological differentiation among species,and these species may be “species on the speciation way”;4)The difference in morphology between S.bodinieri and S.pachyneura is not evident,in contrast to the remarkable genetic difference and phylogenetic context.Morover,the intraspecific variation within both species is high.To facilate the identification,the differences between the two species and the discription of the intraspecific variation are provided.2.S.arenaria complex.The common feature of the members of this complex,all from subg.Saussurea sect.Strictae,are capitula usually in center of leaf rosette and the plants are close in size.This complex contains S.arenaria,S.stoliczkae,S.kansuensis and S.ceterach.The main differences among species are the shape of lobes,the way of pinnately lobed,the type of leaf trichomes and the size of involucre.However,the variability of these traits is relatively large,so there are many intermediate individuals between species.We extensively sampled the complex species and collected 186 individuals from 106 populations,and conducted phylogenetic analysis using ITS and three chloroplast DNA fragments.Our main results and speculations are as follows: 1)The members of the complex form a monophyly on the phylogenetic tree,indicating that the members are possibly a natural group;2)In the nuclear gene phylogenetic tree,four species are clustered into two clades,one consisting of S.arenaria and S.stoliczkae,and the other consisting of S.kansuensis and S.ceterach,but these species on the two clades are not formed a corresponding monophyly,indicating that the genetic differentiation and morphology of the four species were inconsistent;3)Sampling individuals of S.arenaria and S.kansuensis are located in two different clades on the chloroplast phylogenetic tree,which conflict with the results on the nuclear gene phylogenetic tree,We speculate that hybridization is the main cause of this result.3.S.tatsienensis complex.This complex contains S.acutisquama,S.tatsienensis,S.hieracioides and S.superba,which from subg.Saussurea sect.Saussurea,and S.iodostegia and S.nigrescens from subg.Amphilaena.Despite classified into different subgenera,these species are highly similar in terms of stems,leaves and synflorescence,with small differences between species and many intermediate individuals.We investigated 130 individuals from 83 populations representing six species in this complex,and comprehensive morphological characteristics,molecular data,genetic distance,etc.Our main results and speculations are as follows: 1)The resolution of chloroplast genes is very low,and the overall mean distance is only 0.1%,and the interspecific genetic diversity of nuclear ITS is also low,the maximum distance between individuals is only 2.5%,revealing that the species in the complex may be the result of recent radiation evolution;2)Phylogenetic analysis of nuclear genes indicated that S.hieracioides is the earliest differentiated species in the complex,the remaining species are clustered into a clade and contain three subclade with unclear relationship.One consists S.iodostegia and S.nigrescens,the remaining two branches contain S.acutisquama;3)Some morphologically typical S.acutisquama and S.superba clustered into a clade on the nuclear gene phylogenetic tree,with a genetic distance of only 0.1%,indicating that the genetic differentiation and morphological differentiation of the two species are inconsistent,speculating that the individuals of one species is the result of hybridization;4)The intermediate individuals between S.acutisquama and S.tatsienensis is clustered with S.tatsienensis on the nuclear gene phylogenetic tree,with a genetic distance of only0.3%,but the genetic distance from S.acutisquama is 2.5%,so the genetic difference is also inconsistent with the morphological observation.It is possible that these intermediate individuals should belong to the intraspecific variation of S.tatsienensis,but further research is needed.