ABSTRACT Aim To propose and validate a novel integrated framework for the verification and delimitation of cryptic species in widely distributed, morphologically conservative taxa, using the cosmopolitan oligochaete Lumbriculus variegatus as a model system. This framework moves beyond single‐source data by synergistically combining molecular phylogenetics, ecological niche characterisation, historical biogeographic reconstruction and cytogenetic evidence to provide robust, multi‐dimensional validation of evolutionary lineages and their divergence mechanisms. Location Global dataset of 2595 filtered occurrence points with additional sampling across China. Taxon Lumbriculus variegatus (Annelida Clitellata). Methods We developed a ‘phylogeny‐niche modelling’ workflow. (1) Putative evolutionary lineages were first delineated using mitochondrial COI sequences via phylogenetic analysis, haplotype networking and genetic distance calculation. (2) An environmental discriminant model, based on bioclimatic and elevation data from genetically identified samples, was built to ecologically classify all occurrence records into these lineages. (3) For each lineage, optimised MaxEnt ecological niche models were constructed and projected onto the Last Glacial Maximum, the Mid‐Holocene and the current climates to reconstruct historical distribution dynamics. Niche breadth and overlap were quantified. (4) Genome size estimates from flow cytometry and genome surveys provided independent cytogenetic validation of lineage divergence. Results The framework robustly identified two deeply divergent, evolutionarily independent lineages (CI and CII) within L. variegatus . Their inter‐lineage genetic distance (0.158) exceeded conservative species‐level thresholds. While showing complex transcontinental sympatry, the lineages exhibited significant ecological niche differentiation: CI occupies warm‐humid niche, whereas CII has a broader niche adapted to cooler, circumpolar and high‐altitude regions. Historical projections revealed distinct glacial refugia and postglacial expansion pathways. Crucially, a nearly ninefold difference in genome size provided strong independent support for their distinct evolutionary status. From the Last Glacial Maximum to present, niche overlap decreased while geographical overlap increased, indicating niche divergence alongside range expansion. Main Conclusions The study successfully presents and validates a powerful, reproducible framework that integrates phylogenetic, ecological, historical and genomic evidence to resolve cryptic diversity. The application to L. variegatus confirms its efficacy, revealing at least two independent species maintained by ecological niche divergence and major genomic reorganisation. This integrated approach overcomes the limitations of single‐method analyses and provides a generalizable pipeline for species delimitation in taxonomically challenging groups with high cryptic diversity and low dispersal capacity.
Zhou et al. (Sun,) studied this question.