Achene Micromorphology And Biogeography Of Leontopodium In China

Leontopodium R.Brown ex Cass.belongs to the tribe Gnaphalieae in the family Asteraceae;and is mainly distributed in the mountains of Asia and Europe.It comprises approximately 60 species worldwide,of which 37 are distributed in China.It is the second-largest genus of Gnaphalieae in the northern hemisphere.The species of Leontopodium are widely distributed.The characteristics are complex and the species boundary is fuzzy and difficult to distinguish.So the phylogenetic relationship is controversial in Leontopodium.Previous studies have conducted research on the macromorphological characteristics of Leontopodium,but there are few previous studies on the micromorphology of this genus.The achene micromorphology is not easily changed by the environment.There are obvious differences among the Gnaphalieae species,and there are some differences among the species.The achene micromorphology has a high classification value,but it has not been applied to the classification of Leontopodium.In addition,there are also few reports on the biogeographical studies of Leontopodium.Previous studies only involved individual species of Leontopodium,and no systematic research has been conducted yet.In this study,we studied the Chinese Leontopodium from the perspectives of achene micromorphology and biogeography,aiming to find important morphological evidence and evaluate the taxonomic value,and then explore the phylogenetic relationship at the genus and species level,as well as the the biogeographic history of Leontopodium.This study could provide basic data for the biogeographic history of the entire Leontopodium and even the Gnaphalieae in the northern hemisphere.The main results are as follows.1.In this study,the scanning electron microscope(SEM)was used to observe and study the micromorphology characteristics of achenes of Chinese Leontopodium(30 species and 1 variety).The results showed that the achenes of Leontopodium species were elliptical or narrowly elliptical,0.7-1.6-mm long × 0.1-0.6-mm wide.The surface ornamentation was reticulate or rippled with clavate twin hairs or smooth.The carpopodium base was constricted or unconstricted.The characteristics of the achene trichome,surface ornamentation,and carpopodium showed important taxonomic value at different taxonomic levels.(1)The shape of the achene and trichome was valuable for the taxonomic delimitation of taxa between Leontopodium and its related genera.The achenes of Leontopodium were elliptical or narrowly elliptical with clavate twin hairs or smooth.The achenes of its related genera were cylindrical or oblong with globose or papillary twin hairs.(2)The achene surface ornamentation could be categorized into two types(Type ?,reticulate,and Type ?,rippled)and was a taxonomic tool for classification within Leontopodium.The surface decoration of achenes in the Sect.Nobilia is Type ?,and the Sect.Leontopodium is Type ?.(3)The height and diameter of the carpopodium,the carpopodium base shape and trichome density could be used as the basis for classification at the species level within Leontopodium.2.In this study,nrDNA(ETS and ITS)and cpDNA(ndhF,trnL,trnK,psbA,psbK,rbcL,rpl16,rpl32,rpl32-ndhF,rpl32-trnL,trnL-trnF,petA-psbJ,psbA-trnH and psaC-ndhD)were used as molecular markers,using Bayesian inference(BI)and Maximum likelihood(ML)to construct the phylogenetic tree to analyze the phylogenetic relationship of Chinese Leontopodium(23 species and 2 varieties).Combined with divergence time,geographical distribution and morphological characteristics,this study explored the biogeographic history and traits evolution history of Chinese Leontopodium.The results showed that:(1)Chinese Leontopodium was a monophyletic group and could be divided into two clades.One was composed of L.forrestianum(Subg.Paragnaphalium),whose achene surface ornamentation was reticulate with sparse trichomes,and the carpopodium base was unconstricted.The other was composed of species of Subg.Leontopodium.This clade was divided into two Subclades(Subclade 1 and Subclade 2).Subclade 1 was mainly composed of most species of the Sect.Nobilia(L.conglobatum?L.jacotianum?L.junpeianum and L.microphyllum were from the Sect.Leontopodium).The surface ornamentation was reticulate.Subclade 2 was mainly from the Sect.Leontopodium.The surface ornamentation was rippled.The comprehensive results of achene micromorphology and phylogenetic analysis all supported the traditional species classification,that is,Chinese Leontopodium is divided into two subgenuses.The topological structure of the phylogenetic tree constructed by nrDNA and cpDNA was the same on the whole,and only differed in individual clade.(2)Combined with two time calibration point(Tagetes patula and Helianthus annuus,Stoebe muirii and S.aethiopica),the estimated results showed that the Leontopodium stem age was 7.43 Ma BP(95%HPD:6.43-8.38 Ma BP),which indicated Leontopodium originated in the late Miocene.The Leontopodium crown age was 1.93 Ma BP(95%HPD:1.48-2.37 Ma BP).During this period,L.forrestianum first diverged and separated from from others.The divergence time of each section of Leontopodium was after the Pleistocene.At 1.32 Ma BP(95%HPD:1.02-1.62 Ma BP),the remaining taxa of Leontopodium differentiated into two major clades.The crown age of the first clade was 0.98 Ma BP,and the crown age of the second clade was 0.88 Ma BP.(3)The results of the ancestral distribution area reconstruction showed that the common ancestor of Chinese Leontopodium mainly colonized in the China-Himalayan Subregion,and then spread to Taiwan,Northwest China,Central China,and East China.Afterwards,some species spread to Northwest China and North China.(4)The results of ancestral traits reconstruction showed that the ancestral achene of Chinese Leontopodium was reticulate with sparse trichomes.The stem was herbaceous and the leaf base had no sheath.