Genome-wide evidence for cryptic multi-generational introgression between invasive Bursaphelenchus xylophilus and native B. mucronatus

Hybridization between invasive and native species can generate novel genetic variation with ecological and evolutionary consequences, yet introgression often remains cryptic. We investigated natural hybridization between the invasive pinewood nematode Bursaphelenchus xylophilus, the causal agent of pine wilt disease, and native Bursaphelenchus mucronatus in Japan. Using multiplexed inter-simple sequence repeat genotyping by sequencing, we genotyped 342 field-collected nematodes and then conducted pedigree-calibrated analysis using HIest v2.0, based on 48 highly informative diagnostic single-nucleotide polymorphisms (SNPs), followed by ADMIXTURE, complementary NeighborNet and principal component analyses of a broader 2,756-SNP dataset. The results identified nine high-confidence hybrids (2.6%) across two consecutive years (2023-2024), including relatively balanced F1-like individuals and strongly parentally biased advanced backcross-like individuals, demonstrating recurrent, multigenerational introgression. Four hybrids (44.4%) were undetectable using a single-locus diagnostic marker, showing that advanced introgression can escape conventional surveillance. Chemotaxis assays detected no volatile-mediated pre-mating discrimination, whereas post-mating barriers were incomplete and asymmetric. Crosses with B. xylophilus as the maternal parent showed reduced hatchability, particularly with B. mucronatus mucronatus, while reciprocal crosses generally retained higher hatchability, although some showed reduced fecundity. These results provide a mechanistic basis for natural gene flow in a recent secondary contact zone. Because B. mucronatus kolymensis is widespread in Europe and produces viable hybrid offspring with B. xylophilus in both directions, pathogen establishment could create new opportunities for introgression. Our findings reveal cryptic, multigenerational introgression between an invasive forest pathogen and its native congener, highlighting the importance of genomic surveillance for forest disease management and invasion-risk assessment under climate change.