Development of transferable expressed sequence tag-simple sequence repeat (EST-SSR) markers for delimitating two recently diverged gingers endemic to Taiwan

Species delimitation may be difficult for recently divergent species, particularly those with few distinguishable morphological characteristics. Zingiber kawagoii and Z. shuanglongensis are two recently divergent species endemic to Taiwan whose extremely similar vegetative characteristics hinder their distinction during non-flowering seasons. Given their recent divergence, the speciation process and introgressions between these two gingers warrant exploration. However, such studies are hampered by the absence of appropriate molecular markers. To solve this dilemma, we developed 20 transferable expressed sequence tag-simple sequence repeat loci (EST-SSR). Seven highly differentiated loci were further identified for rapid species delimitation. A preliminary test using discriminant analysis of principal components (DAPC) validated the effective discrimination of these EST-SSR loci, while Bayesian clustering analysis (BCA) revealed obvious introgression events between species, particularly on positive-outlier loci. These results imply adaptive introgressions between these species. However, more sampling and further experiments are necessary to validate this inference and resolve questions regarding the introgression and speciation mechanisms. The development of genetic markers in this study provides appropriate experimental conditions and a basis for further research.