DNA barcodes analyses provide insights into species delineation and possible cryptic species in Amentotaxus (Taxaceae)

Accurate species identification and species delineation is crucial for biodiversity conservation and biological research. DNA barcoding is an efficient tool for species identification and delineation, especially when using next-generation DNA barcodes. Amentotaxus is a small genus of coniferous plants, with all six recognized species currently assessed as threatened. However, species delineation within Amentotaxus has not been thoroughly evaluated using DNA barcodes. In this study, we investigated the species discrimination capabilities and delineation using multiple samples per species, employing standard, super-, and next-generation DNA barcodes and their combinations alongside Skmer analysis with genome skimming sequencing data. Our results revealed that the nrDNA cistron exhibited the highest species discriminatory power, successfully resolving five out of the six species. This was followed by ITS, the next-generation barcode (plastome + nrDNA), super-barcode (plastome), and Skmer analysis, each identifying four species. The combinations of the three plastid standard barcodes (rbcL + matK + trnH-psbA) and all four standard DNA barcodes (rbcL + matK + trnH-psbA + ITS) successfully identified only two and three species, respectively. We propose plastid gene spacer clpP-rps12 as an Amentotaxus-specific DNA barcode since it alone delineated four species. Additionally, our results suggested that A. poilanei has recently diverged from A. hekouensis as a budding speciation event. The discordance in the placement of sample AM61 in the nuclear and plastid phylogenetic trees suggests a hybrid origin for this sample, which resulted in the failure to discriminate A. assamica using plastome data. Additionally, the results also imply the potential existence of an undescribed cryptic Amentotaxus species in north Myanmar. Our findings enhance our current understanding of species diversity within Amentotaxus and provide critical insights for conservation efforts aimed at preventing the extinction of the undescribed potentially cryptic species in the wild.