The species-level taxonomy of Eulimnadia has been a subject of concern because of the plasticity observed in adult morphological characters, which often results in low taxonomic resolution. In the past three decades, conservative egg morphology has become the most reliable taxonomic characteristic, especially for the identification of sibling species. To resolve these taxonomic uncertainties and clarify phylogenetic relationships within Eulimnadia cylindrova sensu lato, we integrated morphological analyses (adult soft-body anatomy and egg shell morphology via scanning electron microscopy) and molecular data (four gene markers: nuclear 28S rRNA, mitochondrial 12S rRNA, cytb, and cox1). Species delimitation was performed using distance-based (ABGD) and tree-based (bPTP) methods, combined with maximum likelihood (ML) and Bayesian inference (BI) phylogenetic reconstructions. Our results confirmed the validity of Eulimnadia taoluoensis: it is morphologically distinct from Eulimnadia braueriana and Eulimnadia indocylindrova; notably, it forms an independent clade from E. braueriana in phylogenetic trees, with substantial cox1 (22.34 %) and cytb (14.18–14.90 %) genetic divergences (rejecting prior synonymy hypotheses). Molecular phylogenetic analyses and phylogenetic signal dissection strongly supported that E. cylindrova sensu lato was non-monophyletic and that at least four distinct clades within E. cylindrova sensu lato have been revealed. Notably, E. taoluoensis is nested within the Mexican population of E. cylindrova sensu lato with low cytb genetic divergence (0.55–1.05 %), representing preliminary molecular evidence suggesting potential conspecificity. Future studies incorporating comprehensive morphological comparisons and multi-locus genetic data will be critical to validate the taxonomic status of these closely related lineages. Our findings provided the first molecular data for E. taoluoensis and demonstrated its species validity and the systematic position of E. cylindrova sensu lato. This study demonstrated the utility of integrative taxonomy in resolving taxonomic controversies in groups with morphological plasticity and offered critical insights for species delimination of Eulimnadia.
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