A Comparative Study on the Identification of <scp> <i>Xanthium sibiricum</i> </scp> Patrin ex Widder and <scp> <i>Xanthium italicum</i> </scp> Moretti Based on Three Microscopy Technology

Xanthium sibiricum Patrin ex Widder and Xanthium italicum Moretti are morphologically similar fructus that are frequently misidentified. Xanthium italicum Moretti may possess inherent toxicity, and its adulteration of genuine medicinal materials poses a threat to clinical drug safety. Macroscopic observation and three microscopic techniques including stereo microscope, optical microscope, and 3D X-ray microscope were used for morphological identification of Xanthium sibiricum Patr ex Widder and Xanthium italicum Moretti in this study. 3D X-ray microscopy was applied as a novel tool for non-destructive, high-resolution discrimination of the two taxa. Intact fructus (n = 30 per species) were first screened macroscopically, then examined by stereo microscopy, optical microscopy, and 3D X-ray microscopy (0.3, 0.7, 1.5, 3.5, 18.06, 20.01 μm voxel size, Zeiss Xradia 520 Versa). The results showed that stereo microscopy, optical microscopy, and 3D X-ray microscopy collectively confirm the same conclusion from three distinct physical perspectives: surface topography, internal two-dimensional structure, and internal three-dimensional density distribution. The two Xanthium species differ significantly in burr spine morphology, fructus size and shape, the architecture and distribution of non-glandular and glandular trichomes, cotyledon conformation, and seed-coat cell patterning. In particular, 3D X-ray microscopy clearly resolves internal cotyledon spatial configurations and involucral cavity architectures, which furnishes critical endomorphic characters for taxonomic diagnosis. 3D X-ray microscopy provides unprecedented volumetric contrast of surface spines and internal seed architecture, permitting confident, non-destructive species identification. This study provides a basis for the safe clinical use of Xanthium sibiricum Patrin ex Widder. The frontier of 3D X-ray microscopy in plant systematics offers a novel, rapid, accurate and non-destructive protocol for the discrimination of morphologically elusive species.