Miscanthus sinensis (Poaceae) is typical of many dominant grasses of East Asia. Due to its characteristics of fast growth and high biomass, Miscanthus, a C4 plant, has been long explored for the potential usage as biofuel. In this study, we described the development of nine microsatellite loci from M. sinensis for genetic studies. These new markers were tested in 25 individuals of five populations in three varieties. The number of alleles ranged from 3 to 8. The expected (H E ) and observed (H O ) heterozygosities were 0.37-0.78 and 0.05-0.52, respectively. All microsatellite loci are significantly deviated from Hardy-Weinberg expectations likely due to the population structure within samples. Linkage disequilibrium between four loci pairs were observed from the total 36 pairwise comparisons of loci. Cross-species transferability revealed that all loci can be applied to its closely related species, M. floridulus.

Summary• Outcrossing Arabidopsis species that diverged from their inbreeding relative Arabidopsis thaliana 5 million yr ago and display a biogeographical pattern of interspecific sympatry vs intraspecific allopatry provides an ideal model for studying impacts of gene introgression and polyploidization on species diversification.• Flow cytometry analyses detected ploidy polymorphisms of 2· and 4· in Arabidopsis lyrata ssp. kamchatica of Taiwan. Genomic divergence between species ⁄ subspecies was estimated based on 98 randomly chosen nuclear genes. Multilocus analyses revealed a mosaic genome in diploid A. l. kamchatica composed of Arabidopsis halleri-like and A. lyrata-like alleles.• Coalescent analyses suggest that the segregation of ancestral polymorphisms alone cannot explain the high inconsistency between gene trees across loci, and that gene introgression via diploid A. l. kamchatica likely distorts the molecular phylogenies of Arabidopsis species. However, not all genes migrated across species freely. Gene ontology analyses suggested that some nonmigrating genes were constrained by natural selection.• High levels of estimated ancestral polymorphisms between A. halleri and A. lyrata suggest that gene flow between these species has not completely ceased since their initial isolation. Polymorphism data of extant populations also imply recent gene flow between the species. Our study reveals that interspecific gene flow affects the genome evolution in Arabidopsis.

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These authors contributed equally to this work. SUMMARYYoung incipient species provide ideal materials for untangling the process of ecological speciation in the presence of gene flow. The Miscanthus floridulus/sinensis complex exhibits diverse phenotypic and ecological differences despite recent divergence (approximately 1.59 million years ago). To elucidate the process of genetic differentiation during early stages of ecological speciation, we analyzed genomic divergence in the Miscanthus complex using 72 randomly selected genes from a newly assembled transcriptome. In this study, rampant gene flow was detected between species, estimated as M = 3.36 3 10 À9 to 1.20 3 10 À6 , resulting in contradicting phylogenies across loci. Nevertheless, BEAST analyses revealed the species identity and the effects of extrinsic cohesive forces that counteracted the non-stop introgression. As expected, early in speciation with gene flow, only 3-13 loci were highly diverged; two to five outliers (approximately 2.78-6.94% of the genome) were characterized by strong linkage disequilibrium, and asymmetrically distributed among ecotypes, indicating footprints of diversifying selection. In conclusion, ecological speciation of incipient species of Miscanthus probably followed the parapatric model, whereas allopatric speciation cannot be completely ruled out, especially between the geographically isolated northern and southern M. sinensis, for which no significant gene flow across oceanic barriers was detected. Divergence between local ecotypes in early-stage speciation began at a few genomic regions under the influence of natural selection and divergence hitchhiking that overcame gene flow.

These new genic microsatellite markers are useful for characterizing different traits in breeding programs or to select genes useful for biofuel.