Unravelling phylogenetic relationships within the genus Lispe (Diptera: Muscidae) through genome-assisted and de novo analyses of RAD-seq data

This is the pre-peer reviewed version of the following article: Walczak K., Piwczyński M., Pape T., Johnston N.P., Wallman J.F., Szpila K., Grzywacz A. 2025. Unravelling phylogenetic relationships within the genus Lispe (Diptera: Muscidae) through genome-assisted and de novo analyses of RAD-seq data. Molecular Phylogenetics and Evolution, which has been published in final form at https://doi.org/10.1016/j.ympev.2025.108291.

Lispe represents a species-rich genus within the family Muscidae. The current subdivision of Lispe species into species groups is based mainly on adult morphology and ecology, with the only available phylogenetic study based on three molecular markers. Nonetheless, certain species groups remain unclear, and the relationships and composition of these groups are still unresolved. This study employs restriction-site associated DNA sequencing (RAD-seq) with both reference-based and de novo reads assembly approaches to investigate relationships within Lispe. To apply a reference-based approach we utilised Oxford Nanopore Technologies long read sequencing to assemble a draft genome of L. tentaculata. The resulting topologies of phylogenetic trees are well-supported and relatively consistent, here divided into three main clades. One comprises the palposa-, rigida- and caesia-groups; another includes the nicobarensis-, nivalis-, scalaris- and tentaculata-groups; and the next consists of the longicollis-, desjardinsii-, uliginosa- and kowarzi-groups. The primary discrepancy between topologies obtained under various analytical approaches is the relationship between the leucospila-group and all other ingroup taxa, being a sister taxon either to all remaining Lispe or to a clade consisting of the longicollis-, desjardinsii-, uliginosa- and kowarzi-groups. Lispe polonaise, included for the first time in a molecular phylogenetic analysis, is nested within the caesia-group. Similarly, L. capensis and the hitherto unassigned L. mirabilis belong to the tentaculata-group. Our study confirms the validity of the 14 species groups currently recognised in the genus Lispe.