Evaluation of plastid and nuclear DNA barcode regions for identifying gypsum endemic plants in Semnan Province, Iran

Gypsum ecosystems of the Irano–Turanian region harbor highly specialized endemic plant species that are often geographically restricted and vulnerable to habitat disturbance. Accurate molecular identification of these taxa is therefore important for biodiversity assessment and conservation management. In this study, we evaluated the performance of three DNA barcode regions, including two plastid markers ( rpl32–trnL(UAG) and trnH–psbA ) and the nuclear ribosomal ITS region—for discriminating eight gypsum endemic plant species from Semnan Province, Iran, including Acantholimon cymosum , Astragalus fridae , Astragalus semnanensis , Euphorbia gypsicola , Euphorbia bungei , Gypsophila mucronifolia , Moltkia gypsacea , and Nepeta eremokosmos . Genomic DNA was amplified and sequenced using standard PCR and Sanger sequencing protocols, and barcode performance was assessed based on amplification success, sequence quality, alignment efficiency, and interspecific divergence. Among the tested loci, rpl32–trnL(UAG) showed the highest amplification and sequencing success and provided the greatest discriminatory power among taxa. The ITS region also exhibited substantial interspecific variation and complemented the plastid barcode data for species identification. Although all trnH–psbA sequences were successfully aligned, this marker required more manual refinement because of insertion–deletion polymorphisms and fragment-length variation than the other two loci. Combined analyses of rpl32–trnL(UAG) and ITS enabled reliable discrimination of all studied taxa and suggested preliminary genetic differentiation among geographically isolated gypsum endemic species. Our results demonstrate that integrating plastid and nuclear DNA barcode regions provides an effective molecular framework for documenting and monitoring gypsum specialist flora in arid ecosystems and provides a valuable baseline for future conservation genetic and biodiversity studies in Semnan Province.