Resolving species boundaries in Mediterranean Xiphinema (Nematoda: Longidoridae) through multivariate morphometrics and multilocus profiling: four new species and two cryptic complexes

Abstract As a major biodiversity hotspot, the Mediterranean Basin provides an ideal setting for advancing our understanding of the diversity patterns of plant-parasitic nematodes. The genus Xiphinema is notably diverse in Mediterranean agroecosystems, yet species delimitation remains challenging due to the presence of cryptic lineages. Here, we address this issue by integrating morphology, morphometrics, and molecular data to characterise 50 Xiphinema populations from 264 sampling sites across agricultural landscapes and natural habitats in the main olive-growing regions of the Mediterranean Basin. Our findings yielded four major findings: (i) identification of four new species, X. andalusomarocense sp. nov. X. africana sp. nov. X. alentejanum sp. nov. and X. cryptoduriense sp. nov.; (ii) discovery of two well-supported cryptic complexes by combining multivariate morphometric analyses, multilocus molecular profiling, and phylogenetic inference ( X. baetica- and X. duriense -complexes); (iii) demonstration of the diagnostic value of morphological variation in size and shape for delimiting species within cryptic complexes; and iv) the generation of new ribosomal and mitochondrial sequences for 18 Xiphinema species, expanding the molecular resources available for this group. Together, these results refine the systematics of Xiphinema , broaden our understanding of nematode diversity in the Mediterranean Basin, and provide a robust framework for future evolutionary research on Xiphinema .