| Halenia elliptica D.Don is a diploid biennial herb plant in the Gentianaceae family.It is mainly distributed at elevations ranging from 700 m to 4000 m in southwest,central and north China.There are four spurs on each flower of H.elliptica,providing nectar rewards,and the spur length reduced with the increase of latitude among different populations in the species.Generally,variations of spur length are associated with pollinator shifts,and thus H.elliptica is a suitable plant material for the analysis of mating system evolution correlated with floral traits.In this thesis,field experiments were performed in three populations of H.elliptica,with the aim to find how this species produces seed in the natural environments.Based on de nova transcriptome sequencing,polymorphic SSR markers for H.elliptica were developed.Eleven pairs of SSR markers were employed to analyze selfing rate on population level in seven populations,and the relationships between selfing rate and spur length and latitude were further analyzed.Then,genetic diversity and genetic structure of 16 populations were analyzed with 12 pairs of SSR markers.These results provide an effective evidence for the evolution of mating system and floral traits of H.elliptica.The main results are as follows:1.Pollination ecology of H.ellipticaI investigated mating system,pollinators and selective strength on floral traits in three populations of H.elliptica,respectively as Lijiang population in meadow(LJPM),Huangyuan population in meadow(HYPM)and Huangyuan population in forest(HYPF).No significant difference was found in flowers subjected to hand selfing and outcrossing in each of the three populations,indicating that H.elliptica was fully self-compatible.In conparision with control,seed production of flowers subjected to netting and emasculation was reduced to a certain degree,indicating a mixed mating system of H.elliptica.Seed production of flowers subjected to emasculation suggested that LJPM produced seeds via prodomant outcrossing in comparison with HYPM and HYPF.In contrast,HYPM and HYPF mainly produced seeds via predominant selfing.Bombus sp.and Apis sp.were determined to be the frequent pollinators of H.elliptica.Pollinator visitation frequency in LJPM(0.613±0.062 times/flower/h)was significantly higher than that in HYPM(0.299±0.036)and HYPF(0.239±0.032),but there was no significant difference in the latter two populations.I also measured natural selection on five floral traits(spur length,flower height,flowering time,flower number and flower height to the ground)of H.elliptica via female fitness by comparing emasculated flowers and naturally pollinated flowres in the three populations.In LJPM,significant pollinator-mediated selection was only found on flower height to the ground(?_e=0.099).In HYPM,significant pollinator-mediated selection was only found on spur length(?_e=0.072).In HYPF,significant pollinator-mediated selection on flower height to the ground(?_e=0.085).2.De novo transcriptome sequencing of H.elliptica and development of SSR markerIn this study,the de novo transcriptome for H.elliptica was determined with RNA-seq.A number of microsatellite markers were identified and 126 pairs of SSRs primers were designed for PCR amplification.Twelve SSR markers with polymorphism among populations were found and can be used for future studies in H.elliptica.The SSR markers with polymorphism identified in this study provide valuable genetic resources and represent an initial step for exploring the genetic diversity and population history of H.elliptica.3.Mating system and floral traits of H.ellipticaI investigated the spur length of 7 populations along a latitude gradient,and found that the relationship between spur length and latitude was negative(r=-0.759,P=0.048),indicating spur length of H.elliptica decreased in the population at the high latitudes in comparison with populations at the low latitude.Base on 11 pairs of SSR marker,selfing rate on population level ranged from 0.140 to 0.651,with a mean of0.427.Furthermore,with the increase of latitude,selfing rate increased significantly(r=0.946,P=0.001),but spur length deceased signficantly(r=-0.845,P=0.017),indicating predominant selfing in the population with short spur length at the high latitude.Selfing rate is positively related with inbreeding depression(r=0.935,P=0.002),indicating that indicating that high degree of inbreeding depression might prevent evolution towards complete selfing in populations with short spur length in the high altitude populations,which could maintain mixed mating system of H.elliptica.4.Genetic diversity and genetic structure in populations of H.ellipticaBase on the 12 pairs of SSR markers,each of the 16 populations deviated from Hardy-Weinberg Equilibrium(HWE)significantly,indicating occurrence of genetic drift in all popualtions of H.elliptica.Base on TPM and SMM models,bottleneck effect was only found in QHQL population.Higher levels of PIC(PIC=0.7148>0.5)was found in H.elliptica,but genetic diversity on species level was low(He=0.310<0.61).Furthermore,55%of genetic variations were found among population,suggesting genetic differentiation among populations.The UPGMA and STRUCTURE clustering analysis suggested a close relationship between genetic distance and geographical distance,and this was in consistent with Mantel Test(r=0.403,P=0.030).In addition,the expected heterozygosity(He)and the observed heterozygosity(Ho)are negatively related with latitude(He,r=-0.535,P=0.016;Ho,r=-0.571,P=0.01).Collectively,results of this thesis suggest that H.elliptica could produce seed via autonomous selfing and pollinator mediated outcrossing,indicating a mixed mating system.Evolution towards high frequency of selfing at the high latitude could be an adaptation to infrequent pollinator service in new inhabits.However,the strong inbreeding depression in high latitude population might prevent the evolution towards complete selfing,indicating the importance of mixed mating system and a“better than nothing”role of autonomous selfing in populations with infrequent pollinator service.Variations of spur length could result from the evolution of automous selfing and the associated reduction of resource allocation to flowers in populations at the high latitudes.The mixed mating system of H.elliptica might be helpful in overcoming pollinator scarcity in newly colonized populations towards high latitude after origination in the mountain regions in China.The low genetic diversity was showed on species level,it maybe related to genetic drift,small effective population size and lost alleles,at the same time maybe related to the species in the process of expansion to higher latitudes due to the founder effect reduce genetic diversity. |
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