Delimiting species requires multiple sources of evidence.Here,we delimited two varieties of Halenia elliptica(Gentianaceae)using several lines of evidence,including morphological traits and mating system in a sympatric population,phylogenetic relationships based on nrITS and cpDNA(rpl16)data,and complete chloroplast genome sequences.Comparative analysis of 21 morphological traits clearly sepa-rates the two varieties of H.elliptica.Examination of the flowering process and pollination treatments indicate that H.elliptica var.grandiflora produces seeds via outcrossing,whereas H.elliptica var.elliptica produces seeds via mixed mating.Furthermore,hand-pollinated hybridization of the two varieties produced no seeds.Observations of pollinators showed that when bees began a pollination bout on H.elliptica var.grandiflora they preferred to continue pollinating this variety;however,when they began a pollination bout on H.elliptica var.elliptica,they showed no preference for either variety.Phylogenetic analysis confirmed the monophyly of H.elliptica,which was further divided into two monophyletic clades corresponding to the two varieties.A large number of variants from the chloroplast genomes reflected remarkable genetic dissimilarities between the two varieties of H.elliptica.We recommend that the two varieties of H.elliptica should be revised as two species(H.elliptica and H.grandiflora).Our findings indicate that H.elliptica varieties may have split into two separate species due to a shift in mating system,changes in flowering phenology and/or post-pollination reproductive isolation.

Jin-Feng Wu;Dong-Rui Jia;Rui-Juan Liu;Zhi-Li Zhou;Lin-Lin Wang;Min-Yu Chen;Li-Hua Meng;Yuan-Wen Duan

School of Life Sciences,Yunnan Normal University,Kunming 650092,PR China;School of Ecology and Environmental Science&Yunnan Key Laboratory for Plateau Mountain Ecology and Restoration of Degraded Environments,Yunnan University,Kunming 650091,PR China;Northwest Institute of Plateau Biology,Chinese Academy of Sciences,Xining 810001,PR China;The Germplasm Bank of Wild Species,Institute of Tibetan Plateau Research at Kunming,Kunming Institute of Botany,Chinese Academy of Sciences,Kunming 650201,Yunnan,PR China;Yunnan Lijiang Forest Ecosystem National Observation and Research Station,Kunming Institute of Botany,Chinese Academy of Sciences,Lijiang 674100,Yunnan,PR China

植物多样性（英文）

[1]Jin-Feng Wu;Dong-Rui Jia;Rui-Juan Liu;Zhi-Li Zhou;Lin-Lin Wang;Min-Yu Chen;Li-Hua Meng;Yuan-Wen Duan-.Multiple lines of evidence supports the two varieties of Halenia elliptica(Gentianaceae)as two species)[J].植物多样性（英文）,2022(03):290-299

Gentianaceae,Delimiting,nrITS,pollinating

Multiple,lines,evidence,supports,two,varieties,Halenia,elliptica,species,requires,multiple,sources,Here,delimited,using,several,including,morphological,traits,mating,system,sympatric,population,phylogenetic,relationships,cpDNA,rpl16,data,complete,chloroplast,sequences,Comparative,analysis,clearly,rates,Examination,flowering,process,pollination,treatments,indicate,that,grandiflora,produces,seeds,via,outcrossing,whereas,mixed,Furthermore,hand,pollinated,hybridization,produced,Observations,pollinators,showed,when,bees,began,bout,they,preferred,continue,this,variety,however,preference,either,Phylogenetic,confirmed,monophyly,which,was,further,divided,into,monophyletic,clades,corresponding,large,number,variants,from,genomes,reflected,remarkable,dissimilarities,between,We,recommend,should,revised,Our,findings,may,have,split,separate,due,shift,changes,phenology,post,reproductive,isolation

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