Genetic Differentiation and Delimitation between Ecologically Diverged Populus euphratica and P. pruinosa

Background: The fixed genetic differences between ecologically divergent species were found to change greatly depending on the markers examined. With such species it is difficult to differentiate between shared ancestral polymorphisms and past introgressions between the diverging species. In order to disentangle these possibilities and provide a further case for DNA barcoding of plants, we examine genetic differentiation between two ecologically divergent poplar species, Populus euphratica Oliver and P. pruinosa Schrenk using three different types of genetic marker. Methodology/Principal Findings: We genotyped 290 individuals from 29 allopatric and sympatric populations, using chloroplast (cp) DNA, nuclear (nr) ITS sequences and eight simple sequence repeat (SSR) loci. Three major cpDNA haplotypes were widely shared between the two species and between-species cpDNA differentiation (FCT) was very low, even lower than among single species populations. The average SSR FCT values were higher. Bayesian clustering analysis of all loci allowed a clear delineation of the two species. Gene flow, determined by examining all SSR loci, was obvious but only slightly asymmetrical. However, the two species were almost fixed for two different nrITS genotypes that had the highest FCT, although a few introgressed individuals were detected both in allopatric and sympatric populations. Conclusions: The two species shared numerous ancestral polymorphisms at cpDNA and a few SSR loci. Both ITS and a combination of nuclear SSR data could be used to differentiate between the two species. Introgressions and gene flow were obvious between the two species either during or after their divergence. Our findings underscore the complex genetic differentiations between ecologically diverged species and highlight the importance of nuclear DNA (especially ITS) differentiation for delimiting closely related plant species.