A comprehensive workflow for resolving Gryllidea phylogeny using universal single-copy orthologs

The Gryllidea exhibits remarkable species diversity, yet the higher-level phylogenetic relationships have long been contentious. This study aims to develop and validate a reproducible phylogenetic analysis workflow using Universal Single-Copy Ortholog genes (USCOs) as a scalable genomic backbone to resolve key phylogenetic disputes within Gryllidea. We performed low-coverage whole-genome sequencing (10X) on representative species representing all families of extant crickets. Using an optimized workflow, we performed genome assembly, USCO extraction, and the construction of multiple data matrices with rigorous control of compositional heterogeneity and quality filtering. We reconstructed phylogenies using a multi-model comparison framework. The robustness of key topologies was further assessed through statistical tests including AU, WKH and WSH, and FcLM test. Topology tests consistently and robustly support the division of the Gryllidea into two major clades: Gryllotalpoidea (containing Gryllotalpidae and Myrmecophilidae) and Grylloidea, with internal relationships were resolved as: (Mogoplistidae, (Trigonidiidae, (Phalangopsidae, (Oecanthidae, Gryllidae)))). Our analyses further reveal the non-monophyly of Nemobiinae within Trigonidiidae and clarify the phylogenetic positions of multiple subfamilies. Based on the most reliable topology, divergence time estimates indicate major diversification events began in the Early Jurassic and accelerated during the Cretaceous, coinciding with the rise of angiosperms. Overall, this study provides robust molecular evidence and an evolutionary timescale for resolving long-standing phylogenetic controversies in Gryllidea. Our results emphasize that robust phylogenetic inference requires integrating and comparing multiple complementary datasets and analytical approaches. Within this context, the proposed "matrix optimization-model comparison-topology validation" framework offers a reproducible and extensible foundation for phylogenetic studies.