Species Delimitation Of Two Varieties Of Halenia Elliptica And Reproductive Ecology Of H.elliptica Var.grandiflora

Species is the basic unit of taxonomy,and delimiting species objectively and reasonably is crucial to researches involving biodiversity conservation and sustainable utilization of biological resources.Halenia elliptica(Gentianaceae)is a biennial herb plant,and consist of two varieties,H.elliptica var.elliptica and H.elliptica var.grandiflora,respectively.The two varieties could be distinguished by flower size,spur length and curvature,but a preliminary experiment suggested that artifical hybridization pollination between the two varieties did not produce any seed,indicating reproductive isolation could between them.In order to clarify the classification of H.elliptica var.elliptica and H.elliptica var.grandiflora,we used morphological characteristics,flowering phenology,breeding system,reproductive isolation,pollinator preference,phylogenetic relationships,and chloroplast genome variation to delimit the two varieties.In addition,field survey found that three groups of plants with different sexual systems existed in H.elliptica var.grandiflora,respectively as hermaphrodite plants(only perfect flowers),gynomonoecy plants(perfect flowers and female flowers on same plant)and female plants(only female flowers).To examine the occurrence of differetn sexual systems,field survey was performed in four populations,to investigate the proportion of three groups of plants with sexual systems,differences in plant morphology and flower morphology,and effects of plant size and flowering time on sex expression.The main results are as follows.1.In sympatric populations of two Halenia elliptica varieties,21 morphological traits were measured for each of the two varieties,and the principal component analysis could distinguish two varieties clearly.Field observations on flowering phenology suggested that H.elliptica var.elliptica flowered earlier than H.elliptica var.grandiflora,with flowering peak occurring about 18 days earlier in the former.When flowers of H.elliptica var.elliptica opened,the four anthers began to shed pollen and the two stigma lobes opened immediately,with anthers-stigma contact.In contrast,the four anthers of H.elliptica var.grandiflora began to shed pollen when flower opened,but the two stigma lobes began to open after about two days.Furthermore,stigma was higher than the anthers,indicating protandry and complete herkogamy.Pollination treatments suggested that two varieties were self-compatible.Halenia elliptica var.elliptica could produce seeds via autonomous selfing and pollinator mediated pollination,exhibiting a mixed mating system.In contrast,H.elliptica var.grandiflora relied solely on pollinators to produce seeds,exhibiting outcrossing.Importantly,hand hybridization pollination of the two varieties did not produce any seed,and the reproductive isolation was due to the failure of pollen tube entering micropyle after intrer-variety pollination.Bumblebees and honeybees were the dominant pollinators to the two H.elliptica varieties.By tracing pollinators on different plants,the results showed that bee pollinators would frorage on H.elliptica var.grandiflora when they started their foraging bouts on this variety.However,bees showed no preference to either variety when they they started their foraging bouts on H.elliptica var.elliptica.In addition,based on two markers(nr ITS and rpl16)and the published phenology of Halenia,three populations for each of the two varieties was found to form two monophyletic clades,which corresponded to the two varieties.These results suggested that the two varieties has evolved into two distinct lineages,which could also be supported by the large number of variants from the chloroplast genomes of the two varieties.Taking all results above together,it was suggested that the two varieties of H.elliptica should be raised as two species,and the speciation process was accompanied by changes in the mating system and flowering phenology.2.There were three groups of plants with different sexual systems in H.elliptica var.grandiflora,respectively as hermaprodite,gynomonoecy and female plants.There were significant differences in plant morphology and flower morphology among three groups of plants.There was no significant difference in plant height,leaf length,leaf width,number of lateral branches and total number of flowers between gynomonoecious and female plants,but these traits of the two groups of plants were significantly larger than those of hermaphrodite.Generally,perfect flowers were larger than female flowers.Specifically,perfect flowers on gynomonoecious plants were larger than those on hermaphrodite plants,and female fl owers on female plants were larger than those on gynomonoecious plants.Size-dependent reproduction was found in each of the three groups of plants of H.elliptica var.grandiflora,suggesting a threshold for the beginning of reproduction.The threshold value was largest for gynomonoecious plants.The flowering order of the three sexual plants was different in the H.elliptica var.grandiflora population.For gynomonoecious plants,the female flowers appeared at the end of the flowering period,which might be strongly related to resource limitation by adusting resouce allocation to female function.In summary,two varieties of H.elliptica is supported to be raised to species level based on multiple lins of evidence from morphological traits,phylogenetic relationships,chloroplast genome,mating system and reproductive isolation.We recommend that the two varieties of H.elliptica should be revised as two species,named Halenia elliptica D.Don and Halenia grandiflora(Hemsl.)M.Y.Chen,respectively.There are three groups of plants with different sexual system in H.grandiflora,and reproductive allocation of each group of plants is size-dependent.The polymorphism of plant sex expression is the result of long-term adaptation to the environment,and plastic sex expression might improve reproductive fitness in the high altitude.