| Natural hybridization may slow down or reverse species differentiation by forming gene flow and adaptive introgression,and also it may accelerate speciation through recombination or polyploidization.The Asina Buddleja species(Scrophulariaceae)are young taxa differentiated during the uplift of the Tibetan Plateau.Many Buddleja species in Tibetan Plateau are sympatrically distributed with overlapping florescence,and the interspecific hybridization occurs frequently.Based on the field investigation of geographical distribution,the morphological characters,the palynology,the reproductive biology,molecular biology of B.alternifolia,B.crispa and B.× wardii were comprehensive studied for verifying the presumed natural hybridization origin of B.× wardii.Meanwhile,the potential geographical distribution of B.alternifolia and B crispa in different historical periods were predicted by using the species distribution model.The main results are summarized as follows:1.Geography distributionsThe field investigation on the geographic distribution of B.× wardii,B.alternifolia and B.crispa indicated that these three species has nine sympatric sites,and one sympatric site of B.alternifolia and B.× wardii in Tibetan Plateau.There was no site distributed only B.× wardii or the sympatric site of B.crispa and B.× wardii.In the 9 sympatric distribution sites,B.alternifolia,B.crispa and B.× wardii were found mixed,and there was no obvious geographical isolation among the 3 species.In addition,no other species of Buddleja were found during the field investigation in the same region.Besides two sites of YG and KR,in seven sympatric sites were observed more individuals of B.alternifolia than that of B.crispa and B.× wardii.All the three Buddleja species are mainly distributed on the roadside,the ridge and other anthropogenic disturbance places2.MorphologyThe morphological characters of the three taxa collected from 5 sympatric sites were analyzed,and the results showed that morphologies of B.alternifolia and B.crispa are obviously distinct,and morphological characters of B.× wardii were intermediate between B.alternifolia and B.crispa.Moreover,the morphological diversity of B.alternifolia was lower than B.crispa.The leaf trichomes of B.alternifolia are 8-arms stellate hairs and scattered on the upper surface but densely covered on the lower surface.Both leaf sides of B.crispa are densely covered with 4-arm candelabra hairs.Stellate hairs and candelabra hairs are densely in B.× wardii.The pollen of B.alternifolia,B.crispa and B.× wardii are all subprolate,three colporate,membrane smooth and indistinct.The pollen size of B.alternifolia and B.crispa are distinct with B.alternifolia is smaller(15.43-17.71 ?m,mean 16.70±0.62?m)than B.crispa(16.20-24.32 ?m,mean 20.49±1.82 ?m).The pollen size of B.×wardii is intermediate between B.alternifolia and B.crispa(14.04-19.17 ?m,mean 17.51±1.39 ?m),Moreover,it is significantly different from B.crispa,but not from B alternifolia.The reflectance spectrum of the petals showed some variation between the Buddleja species.B.crispa and B.× wardii were a clearly marked peak in the reflectance spectrum at 485 nm.However,there was no obvious peak in the reflectance spectrum in B.alternifolia.It could be speculated that butterfly(Lepidoptera)and bee(Hymenoptera)might be pollinator for B.crispa and B.× wardii.3.Hand pollination and seed germinationThe hand pollination experiments between B.alternifolia and B.crispa were carried out,and different fruit set and seed germination rates were observed.For the intraspecific of B.alternifolia(?)from Tibetan Plateau × B.crispa(?),the rates of fruit set and seed germination are 6.45%and 0,respectively.On the contrary,the rate of fruit set and seed germination of B.crispa(?)× B.alternifolia(?)from Tibetan Plateau are 64.71%and 61.9%.For the intraspecific of B.alternifolia(?)from the Loess Plateau × B.crispa(?),the rates of fruit set and seed germination are 84.62%and 16.07%,respectively.On the contrary,the rates of fruit set and seed germination of B.crispa(?)× B.alternifolia from the Loess Plateau(?)are 71.88%and 47.2%.Thus,there was no complete reproductive isolation and asymmetric cross compatibility between B.alternifolia and B.crispa.4.Molecular BiologyIn total,96 individuals of three Buddleja species from two populations(BH and TJ),four nuclear loci and three chloroplast intergenic spacers were used to test the natural hybridization between B.altenifolia and B.crispa,and to detect the natural hybridization direction.Most individuals of B.× wardii showed superposition of the peak value of B.altenifolia and B.crispa at the fixed variation sites.The haplotype network showed that most individuals of B.× wardii had haplotypes from both B.altenifolia and B.crispa.The Structure analysis showed that,all but BHWI9,the probability of hereditary substance from B.altenifolia and B.crispa was approximately equal.The Newhybrid analysis showed that B.× wardii were F1 generation.The cpDNA analysis suggested that B.crispa was the maternal parent in BH population.However,the three species in TJ population shared a same haplotype,and direction of the natural hybridization could not be distinguished.5.Genetic diversity and genetic structureTen of microsatellite loci were applied to analyze the genetic diversity and genetic structure of 393 individuals of B.crispa,B.alternifolia and B.× wardii in 9 hybridization sites.The results showed that B.crispa and B.× wardii had high level of genetic diversity with the mean expected heterozygosity(HE)of 0.551 and 0.513 respectively,while the genetic diversity of B.alternifolia was lower,and the mean expected heterozygosity(HE)was 0.380.The lower genetic diversity with large population size of B.alternifolia in many populations might be caused by its cloning characteristics.The FST among different populations of B.alternifolia was 0.253,which was greater than that of B.crispa(FST=0.169),indicating that low gene flow among different populations of B.alternifolia.The genetic differentiation between B.alternifolia and B.crispa was higher,with the mean FST value of 0.416.The PCoA analysis also showed that the genetic structure of both B.alternifolia and B.cripsa populations was clear.The mean FST value among B.× wardii and B.alternifolia or B.crispa was 0.266 and 0.270,respectively,indicating B.× wardii and the two parental species are closely related.The Structure analysis revealed that the proportion of genetic materials from B.crispa and B.alternifolia was continuous in B.× wardii individuals.6.Potential geographical distribution patterns of B.alternifolia and B.crispa in different periodsThe Maximum entropy model(MaxEnt)was used to analyze the potential distribution of B.crispa and B.alternifolia under past(LIG,130 ka years ago;LMG,22 ka years ago;Mid Holocene,6 ka years ago),Current(1970-2000)and future(2050 and 2090)climate conditions.The results showed that the dominant climatic factors affecting the two species distribution were the mean temperature of coldest quarter and the precipitation of driest/wettest quarter.During the LGM,distribution range of B alternifolia was decreased sharply,while distribution of B.crispa was expanded.In the mid Holocene,the two species had overlapping distribution regions in Tibetan Plateau,and then the overlapping distribution range was expanded.Prediction of potential suitable distribution of the Buddleja species in 2050 and 2090 indicated that the suitable habitats will be expanded and distribution center will move to the higher latitude or higher altitude under different greenhouse gas emission scenarios.Along with the increasement of greenhouse gas emissions,the overlapping distribution of B.alternifolia and B.crispa will also be increased,and thus,the natural hybridization zone of the two Buddleja species will be enlarged. |
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