DNA barcoding and phylogenetic relationship in Pakistani species of Aveneae-type plastid DNA clade (Pooideae, Poaceae) based on nuclear and chloroplast DNA markers

The Aveneae-type plastid DNA, clade belonging to the tribe Poeae s.l within the subfamily Pooideae (Poaceae), includes economically, nutritionally and ecologically important grasses. However, due to morphological similarity and prevalence of polyploidy, species boundaries and phylogenetic relationship among its taxa remain uncertain. This study aimed to evaluate the species discrimination power of three universal plant DNA barcode loci (ITS, matK and rbcL) in 20 representative species of the Aveneae-type plastid DNA clade. Genetic distances were computed using MEGA-X software, while tree based maximum likelihood (ML) analyses were performed to infer the phylogeny. On the basis of percent discrimination rate at a significance level of p < 0.05 matK and rbcL barcodes were 1.5% more successful than non-coding ITS region. However, in terms of barcode gap ITS marker showed highest barcode gap (0.075), than matK (0.029), and rbcL (0.008). Maximum Likelihood (ML) tree-based analyses identified 82.35% species using rbcL, 80% species with matK, and 43.75% species with ITS marker. Most subtribes and genera, except Agrostis and Phalaris, formed well-supported monophyletic clades (bootstra p > 90%). This study endorses the effectiveness of all the three barcode regions and provide novel DNA sequences of previously unsampled Himalayan grasses for global Poaceae barcoding initiatives and phylogenetic research.