Study On The Phylogeny And Biogeography Of Gymnospermium

Gymnospermum Spach is an early spring flowering perennial herb in Nandinoideae,Berberidaceae,including about 11 species.They disjunctively distributed in Eurasia of north temperate zone,and 3 species in China.The plants of Gymnospermum have beautiful flowers and many species have medicinal values.They have great ornamental and economic development prospects in future.In addition,most of Gymnospermum species have been included in the international or national rare and endangered plants.However,there are still some controversies about the number of species and interspecific relationships.It was still uncertain about the basical taxa,the phylogeny and spread routes of this genus.To resolve these issues,a strong and highly supportive phylogenetic tree,and further biogeographical reconstruction are urgently needed.Ten Gymnospermum species across the distribution of this genus in this study were sampled.To explore the phylogenetic relationship,biogeography and their classification within the genus and provide a reference for the development,utilization,and protection,we conducted comparative morphology,comparative plastome and their phylogeny,and constructed the phylogenetic trees,divergence time tree,and ancestral distribution regions based on single-copy orthologous genes from transcriptome data of Gymnospermium.The main results were as follows.(1)Through the comparation of the 32 qualitative and quantitative characteristics of 10 Gymnospermum species,the results showed that there are greater variations within species than those intra-species in these characteristics,including plant height,stem height and thickness,leaflet size,total petiole length,small petiole length,inflorescence axis length,number of floret,bract size,floret size,petal shaped sepal size,degenerated petal size,stamen and pistil length,ovary and ovule size.The mature leaves of G.microrhychum distributed in Northeast China are usually a bipinnately compound leaf(with 3 final leaflets and 1 or 2 leaflets split on both sides),while those of G.kiangnanense distributed in East China are usually tripinnate compound leaf(with 3 final leaflets and multiple leaf tips).The two species are easily distinguished from other species.These Gymnospermium species,including two species distributed in Western Tianshan G.altaicum in Altai Mountains,G.smirnowii in Caucasus Mountains,and four spcies in Europe usually have mature leaves of bipinnately compound leaf,with 4-6 small leaves,and there were no significant differences between species.(2)A total of 17 plastomes of 10 Gymnospermum species were assembled,the sequence length ranged from 157,818 bp in G.scipetarum to 161,406 bp in G.smirnovii(I).All the plastomes were highly consistent in gene order.We found a severe truncation(ca.1 KB)at the 5’ end of ycf1 in three G.altaicum plastomes,and a premature termination of rop C1 in G. microrrhynchum due to the insertion of one base,which made it 700 bp shorter than the other plastomes.All the Nandinoideae plastomes were applied as outgroups to infer the phylogeny of Gymnospermum.It produced a phylogenetic tree with high support(only 2 nodes with less than 100% support).we identified G.microrrhynchum from northeastern Asia as the earliest diverging species(Clade A),followed by G.kiangnanense from eastern China(Clade B), while the rest species clustered into the two sister clades(C and D).Clade C included three species from West Tianshan(G.albertii,G.darwasicum,G.vitellinum),and an individual of G.vitellinum was embedded in G.darwasicum.Clade D consisted of G.altaicum from northern Central Asia,plus one species from the Caucasus Mountains(G.smirnovii)and three from southeastern Europe(G.odessanum,G.peloponnesiacum,G.scipetarum).Furthermore, we identified 21 highly variable plastome regions from Nandinoideae with nucleotide diversity(Pi)values > 0.02.(3)In this study,the transcriptomes of 17 individuals from ten species of Gymnospermum and two species of Nandinoideae were assembled,and 685 single-copy orthologous gene(SCOGs)were obtained.The phylogenetic topologies of Gymnospermum based on 685 SCOGs were similar with the phylogenetic relationships based on plastomes.The basal and subbasal taxa still were G.microrrhynchum and G.kiangnanense,Clade C distributed in West Tianshan was embedded in clade D,and one individual of G.odessanum was embedded in two G.peloponnesiacum individuals.Divergence times and ancestral distribution were inferred based on 230 SCOGs.The results indicated that the genus Gymnospermium divided from Leontice17.9 Mya.G.microrrhynchum and G.kiangnanense diverged at 15.86 Mya and 14.67.Mya,respectively,and the diversification time of the remaining species were within 10 Mya.(4)Combined with results of morphologies,molecular phylogenetic trees and geographic distributions,the two species,G.microrrhynchum(Clade A)and G.kiangnanense(Clade B),with long divergent time and significantly different habitats,were easily distinguished in morphology and molecular systematics.The species distributed in Western Tianshan(Clade C),Altai Mountains,Caucasian,and European(clade D)were insignificant differences.However,those two clades has changed phylogenetic position in the plastome tree and the SCOG tree.It was likely that clade C and clade D have undergone a hybridization events or incomplete lineage differentiation.Furthermore,the phylogeny of plastome showed that G.vitellinum embedded in two individuals of G.darwasicum.Plus with no significant morphological differences between the two species,we proposed to merge the two species.The phylogeny based SCOG showed that G.dessanum embedded in the two G.peloponnesiacum individuals,we also proposed to merge the two species combined with their morphological characteristics.(5)The ancestral distribution areas of Gymnospermum maybe were northeast Asia,eastern China and Europe.We suggested Gymnospermum was widely distributed in the northern temperate zone of Eurasia before the species split in the Miocene.Possibly due to the dramatic global climate change during the middle-late Miocene,the species of Gymnospermium expanded southward and discontinuously distributed in Eurasia.Our study further supports the biogeographical hypothesis that Berberidaceae was once widely distributed in the northern temperate zone.