Studies Of Reproductive Isolations Between Related Pedicularis Species

Investigations of reproductive isolation mechanisms are essential for an understanding of species diversification,particularly in plant lineages that have undergone rapid radiation,which diverse species have differentiated in a relatively short period.Determining the role of different types of reproductive isolation barriers and their relative contribution to total reproductive isolation is a major challenge for us to better understand the speciation.Pedicularis,a superdiverse genus with more than 300 species in the Hengduan Mountains,is a monophyletic group of rapid radiation in the North Temperate Zone.Pedicularis species are characterized by diverse corolla morphology,and are effectively pollinated by bumblebees.The reproductive isolation in this genus was studied in some sympatric species.To further understand the speciation of this genus,we used closely related species as experimental system,using the unified calculation method to quantify the reproductive isolation strength and to analyze the importance between pre-zygotic and post-zygotic isolation,including section Longiflorae(Pedicularis cranolopha and P.tricolor),section Cyathophora(P.rex,P.superba and P.cyathophylla)and two sympatric nectared species(P.rex and P.densispica)which were shared the same bumblebee pollinators.The main results are summarized below.(1)Reproductive isolation between P.cranolopha and P.tricolor,two sister species of section Longiflorae.They were similar in floral shape,with long corolla tube and elongated curved beak,and distributed in allopatry.Results of translocation experiments showed that flowering phenology of two species partly overlapped and bumblebee pollinators did not discriminate between flowers of two species,and interspecific pollination was traced by fluorescent powder.Observations of pollen tube growth showed that heterospecific pollen did not perform worse than conspecific pollen.Interspecific hybridization produced viable seeds.Geographical isolation was an important prezygotic reproductive barrier,contributing greater than other barriers to total reproductive isolation.This study suggests effects of geographical isolation on species diversification,giving a clue to understand the rapid radiation on mountainous areas.(2)Reproductive isolation among three closely related species of section Cyathophora.To understand the isolation barriers of related species,three species pairs between the three species were studied:P.rex × P.superba,P.rex × P.cyathophylla and P.superba × P.cyathophylla.Flowering phenology of three species was partially overlapped and all three species shared same bumblebee pollinators at each site.Floral constancy was very high for each pair of species,and bumblebee visited flowers with few interspecific moves.Few heterospecific pollen grains were deposited on the stigmas,and conspecific pollen grains were deposited on different sites of bumblebees' body in species pairs of P.rex and P.superba.Heterospecific pollen could germinate and grow to ovary in 24 hours after pollination treatments,but performed worse than conspecific pollen.Hybridization between species in species pairs produced few seeds.All pre-zygotic reproductive barriers can completely isolate each species from each other.The results suggested that the complete reproductive isolation involved multiple barriers acting together in preventing gene flow.Our study also showed that pre-zygotic isolation contributed more than post-zygotic isolation.(3)Reproductive isolation between two nectared co-flowering species.We investigated flowering phenology,pollinator visits,heterospecific pollen transfer,pollen tube growth and seed set of P.densispica and P.rex.Flowering period of two species partially overlapped but peak flowering periods were separated.Pollinator observation showed that they shared bumblebee pollinators with nototribical pollination behavior and high floral constancy,and bumblebees visiting one species rarely moved to another.Percentage of heterospecific pollen deposition on each species stigma was low,about 2.43%(pollen number 2.3 ± 1.1,mean±SE)and 8.02%(3.6 ± 0.8)of total stigmatic pollen loads of P.densispica and P.rex,respectively.Hand pollination experiments showed that interspecific pollen could germinate on the stigma but few could grow into ovary.No hybrid seeds were produced and the post-zygotic isolation was completely for both two species.Our results suggested that the pre-zygotic isolation mediated by flowering phenology and pollinators was a key barrier to minimize heterospecific pollen transfer and could basically achieve complete isolation between the two species.Post-zygotic isolation may further supplement the effect of pre-zygotic isolation barriers to prevent gene flow between species to maintain species coexistence.Our comprehensive assessment of reproductive isolation in related Pedicularis species showed that both sympatric and allopatric species shared bumblebee pollinators,and temporal(flowering period)and spatial separation(geography)were important barriers in reducing the interspecific reproductive interference to achieve the pre-zygotic isolation.The diversity of hetergeneous habitats on the mountainous areas may have contributed to the rapid differentiation of flowering species.