| Although speciation occurs over a long period of time,it is often initiated by the adaptive divergence of populations occupying ecologically distinct environments.Genetic differentiation is one of the main strategies for organisms to cope with heterogeneous habitats.Genetic and epigenetic variations within and among heterogeneous habitats provide materials for natural selection and adaptive evolution.Elucidating the underlying genetic and epigenetic mechanisms of phenotypic variation within and among heterogeneous habitats and their relationships with adaptive evolution is one of the central topics in molecular ecology.However,little is known about this aspect of study on a small spatial scale.Phragmites australis(Cav.)Trin.ex Steud.is virtually cosmopolitan and is distributed widely across the wetland,sandy soil and grassland of the Songnen Steppe.This species is not only a major forage grass for herbivory during the period of vegetative growth but also a primary candidate for ecological restoration of saline-alkali meadows.We conducted field survey,lab experiment,AFLP and MSAP marker analysis and common garden experiment with Phragmites australis and Phragmites hirsuta in heterogeneous habitats at small scales in the Songnen Steppe.Using statistical analysis of molecular ecology,we aim to explore the genetic and epigenetic basis of phenotypic variation among heterogeneous habitats and their contribution to adaptive evolution.The main results are as follows:(1)Various morphological traits of reed differed significantly among the 4 habitats with remarkably different soil characters.The growth traits concerning height,leaf and biomass were much greater in P.australis from the seasonally waterlogged,low-lying meadow with a pH of 8–8.5(Habitat 1,designated H1)and sandy soil habitat(Habitat4,designated H4)than from the Leymus chinensis+Phragmites australis community(Habitat3,designated H3)and alkaline patch which lacked accumulated rainwater and had a pH greater than 10(Habitat 2,designated H2).In most cases,those traits from H2 were the lowest among the habitats.The stem fraction from H1 and H3 were larger than those from the other habitats.Likewise,the leaf fraction from H4 and the leaf sheath fraction from H2 were larger than the corresponding values from the other habitats.The growth parameters were positively correlated with soil moisture,content of soil total phosphorus,total nitrogen.The stem fraction was positively correlated with those soil traits and organic matter while the leaf fraction was negatively correlated with those traits.The growth parameters were negatively correlated with soil pH,electric conductivity(EC),content of soil NO3-nitrogen,Cl-,SO42-,Na+ and K+.The stem fraction initially increased and then decreased,while the leaf fraction initially decreased and then increased and the leaf sheath fraction constantly increased with increasing EC,content of soil Cl-?Na+ and K+.The growth parameters were positively correlated with content of soilMg2+ and Ca2+.There was evidence of trade-offs between leaf fraction and stem fraction.The correlations between the quantitative characters,biomass allocations as well as allometric partitioning in biomass differed significantly among the 4 habitats.(2)We found genetic polymorphism at the intra-habitat level and intra-population level on a small spatial scale.P.australis in H1 and H3 harbored a higher level of genetic diversity than those in H2 and H4,which was congruent with the niche-width variation hypothesis.The genetic differentiation between alkaline soil habitat(H1,H2 and H3)and sandy soil habitat(H4)was very remarkable.Thirty-three outlier loci under natural selection that correlate with environmental variables,mainly related with content of soil Ca2+,total phosphorus,Mg2+ and soil pH,were detected by modified FDIST,Bayescan and Sam?ada analyses.The results of the present study supported the so-called ‘divergence-with-gene-flow' model.The experiment provided the necessary data at a genome-wide level to help to further our understanding of local adaptation.(3)About two-thirds of the loci from the MSAP analysis were methylation-susceptible loci(MSL).The proportions of the 4 different methylation patterns differed among the heterogeneous habitats.We found relatively high levels of epigenetic diversity at the intra-habitat level and intra-population level and P.australis in H1,H2 or H3 harbored a higher level of genetic diversity than those in H4.Our results implied significant epigenetic differentiation among habitats,especially between alkaline soil habitat(H1,H2 and H3)and sandy soil habitat(H4).There were greater epigenetic than genetic differentiation between H1 and H3 and between H2 and H3 as well.Genetic variation was correlated with epigenetic variation significantly.Epigenetic differentiation among habitats was correlated with adaptive genetic divergence.Correlation analyses revealed that a greater portion of the phenotypic variation was attributed to differences of epigenetic diversity among sites rather than those of genetic diversity.The genetic diversity parameters were correlated with content of soil total phosphorus,Cl-and Ca2+ significantly,while the epigenetic ones were correlated with soil moisture,content of soil total phosphorus,total nitrogen and organic matter significantly.The epigenetic diversity was affected more by soil nutrient availability.(4)Molecular analysis of Phragmites australis,Phragmites hirsuta,Phragmites japonica based on AFLPs were performed using UPGMA,STRUCTURE and Principal Coordinate Analysis.The results showed that Phragmites australis from H3 and H4 differentiated markedly and Phragmites hirsuta from H3 and H4 also differentiated obviously.Phragmites australis in Nanhu Park and Phragmites australis from H3 and H4 were grouped together.The differentiation of Phragmites australis and Phragmites hirsuta was higher than the differentiation due to geographical distance or ecological habitats.We also found clear epigenetic differentiation between Phragmites australis and Phragmites hirsuta based on MSAPs and the individuals from H3 and H4 gathered seperatly within each group.The resultsindicated that the epigenetic differention of Phragmites australis and Phragmites hirsuta on the basis of DNA methylaton was higher than the differentiation due to heterogeous habitats.These findings suggest that Phragmites hirsuta is at early or middle speciation stage when it diverged from Phragmites australis.(5)Field experiment showed that concerntrations of Chla,Chlb,Chl(a+b),Car and the Chl(a+b)/Car ratio decreased significantly in H2(alkaline patch)compared with H1.Only the Chla/Chlb ratio showed no-alkali salt dependent trend in P.australis.The common garden experiment indicated that local adaptation of Chla,Chlb,Chl(a+b),Car concerntrations can be maintained over small spatial scales.Additional support for this inference was provided by the population-genomic approach that detected outlier loci via FDIST,Bayescan and Bayenv,and QST–FST comparisons.Soil moisture,pH and electric conductivity may have acted as selective agents to produce such an adaptive divergence.These findings not only provided insights into genetic and epigenetic basis of P.australis with different phenotypes in heterogeneous habitats at small scales,but also facilitated the study of molecular ecology in reed.The results of the present study may provide insight or guidelines for studies of ecological adaptation,stress resistance,species delimitation of the genus Phragmites and for utilization practices,including ecological revegetation,maintenance of these plant genetic resources and genetic improvement. |
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