This study was conducted as a preliminary investigation to establish a standardized DNA barcoding method for the forensic identification of pteridophyte-derived evidence, including ferns, algae, and plant fragments collected from crime scenes. In forensic investigation, trace plat materials adhering to clothing, footwear, vehicles, or tools can serve as important associative evidence linking individuals, objects, and location. However, conventional morphology-based identification is often limited by the minute size, degraded condition, and lack of diagnostic features in forensic samples. These limitations are particularly pronounced in pteridophytes because of morphological similarity among related taxa, variation across life stages, and the frequent recovery of spores or plant fragments. Therefore, this study evaluated the applicability of DNA barcoding as a reliable method for species-level identification of non-human botanical evidence in forensic contexts.

To enhance forensic applicability, three commercial DNA extraction kits were comparatively assessed in terms of DNA yield and purity from trace plant samples. Among them, the DNeasy Plant Mini Kit showed the highest overall performance with respect to DNA concentration and purity, whereas the Maxwell FSC DNA IQ Casework Kit offered practical advantages for forensic casework, including automated processing, reproducibility, and suitability for handling multiple samples with reduced contamination risk. In parallel, ten candidate barcode loci, including trnH-psbA, atpF-aptH, rbcL, ITS psbK-psbI, and matK, were evaluated for amplification efficiency and taxonomic discriminatory power.

The results demonstrated that single-locus analysis was insufficient for consistent and accurate species identification across diverse pteridophyte taxa. In contrast, multi-locus approaches yielded significantly higher identification success rates and improved analytical reliability. There finding indicate that the combined use of barcode markers can overcome the limitations of individual markers, including locus-specific amplification bias and insufficient taxonomic resolution.

Overall, this study provides foundational experimental evidence for the forensic application of DNA barcoding to trace pteridophyte materials. Nevertheless, additional research is needed to broaden its application to pteridophytes, particularly through the establishment of comprehensive reference databases, the assessment of additional barcode regions, and the validation of analytical performance under diverse forensic sample conditions.

Authors

• EunYoung Park (Criminal Investigation Command, Ministry of National Defense, South Korea)
• HyeJin Lee (Criminal Investigation Command, Ministry of National Defense, South Korea)
• SiNae Han (Criminal Investigation Command, Ministry of National Defense, South Korea)
• JiYoung Kim (Criminal Investigation Command, Ministry of National Defense, South Korea)