Robber or assassin flies (Asilidae) are a diverse family of venomous predators. The most recent classification organizes Asilidae into 14 subfamilies based on a morphological phylogeny, but many of these are not supported by molecular data. To test the monophyly of various clades in Asilidae, we used the recently developed Diptera‐wide ultraconserved element bait set to compile seven datasets comprising 151 robber flies and 145–2496 loci. We also compared the performance of various nodal support metrics. Our Maximum Likelihood phylogeny was fully resolved and well supported, but partially incongruent with the coalescent phylogeny. Further examination of datasets suggested that GC bias had influenced gene tree inference and subsequent species tree analyses. The subfamilies Brachyrhopalinae, Dasypogoninae, Dioctriinae, Stenopogoninae, Tillobromatinae, Trigonomiminae and Willistonininae were not recovered as monophyletic. The inter‐subfamily relationships are summarized as follows: Laphriinae and Dioctriinae (in part) successively sister to the remaining subfamilies, which form two clades: the first consists of a grade of Stenopogoninae (in part), Willistonininae (in part), Bathypogoninae+Phellinae, Stichopogoninae, Leptogastrinae, Ommatiinae and Asilinae; the second clade consists of a paraphyletic assemblage of genera from Dioctriinae (in part), Trigonomiminae, Stenopogoninae (in part), Tillobromatinae, Brachyrhopalinae and Dasypogoninae. This phylogeny demonstrates that the higher classification of Asilidae is far from settled, but does provide a much‐needed foundation for a thorough revision of the subfamily classification.

Robber flies are an understudied family of venomous, predatory Diptera. With the recent characterization of venom from three asilid species, it is possible, for the first time, to study the molecular evolution of venom genes in this unique lineage. To accomplish this, a novel whole-body transcriptome of Eudioctria media was combined with 10 other publicly available asiloid thoracic or salivary gland transcriptomes to identify putative venom gene families and assess evidence of pervasive positive selection. A total of 348 gene families of sufficient size were analyzed, and 33 of these were predicted to contain venom genes. We recovered 151 families containing homologs to previously described venom proteins, and 40 of these were uniquely gained in Asilidae. Our gene family clustering suggests that many asilidin venom gene families are not natural groupings, as delimited by previous authors, but instead form multiple discrete gene families. Additionally, robber fly venoms have relatively few sites under positive selection, consistent with the hypothesis that the venoms of older lineages are dominated by negative selection acting to maintain toxic function.

Robber flies or assassin flies (Diptera: Asilidae) are a diverse family of venomous predators. The most recent classification organizes Asilidae into 14 subfamilies based on a comprehensive morphological phylogeny, but many of these have not been supported in a subsequent molecular study using traditional molecular markers. To address questions of monophyly in Asilidae, we leveraged the recently developed Diptera-wide UCE baitset to compile seven datasets comprising 151 robber flies and 146 - 2,508 loci, varying in the extent of missing data. We also studied the behavior of different nodal support metrics, as the non-parametric bootstrap is known to perform poorly with large genomic datasets. Our ML phylogeny was fully resolved and well-supported, but partially incongruent with the coalescent phylogeny. Further examination of the datasets suggested the possibility that GC bias had influenced gene tree inference and subsequent species tree analysis. The subfamilies Brachyrhopalinae, Dasypogoninae, Dioctriinae, Stenopogoninae, Tillobromatinae, Trigonomiminae, and Willistonininae were not recovered as monophyletic in either analysis, consistent with a previous molecular study. The inter-subfamily relationships are summarized as follows: Laphriinae and Dioctriinae (in part) are successively sister to the remaining subfamilies, which form two clades; the first consists of a grade of Stenopogoninae (in part), Willistonininae (in part), Bathypogoninae+Phellinae, Stichopogoninae, Leptogastrinae, Ommatiinae, and Asilinae; the second clade consists of a thoroughly paraphyletic assemblage of genera from Dioctriinae (in part), Trigonomiminae, Stenopogoninae (in part), Tillobromatinae, Brachyrhopalinae, and Dasypogoninae. We find that nodal support does not significantly vary with missing data. Furthermore, the bootstrap appears to overestimate nodal support, as has been reported from many recent studies. Gene concordance and site concordance factors seem to perform better, but may actually underestimate support. We instead recommend quartet concordance as a more appropriate estimator of nodal support. Our comprehensive phylogeny demonstrates that the higher classification of Asilidae is far from settled, and it will provide a much-needed foundation for a thorough revision of the subfamily classification.

Robber flies are an understudied family of venomous, predatory Diptera. With the recent characterization of venom from three asilid species, it is possible for the first time to study the molecular evolution of venom genes in this unique lineage. To accomplish this, a novel whole-body transcriptome of Eudioctria media was combined with 10 other publicly available asiloid thoracic or salivary gland transcriptomes to identify putative venom gene families and assess evidence of pervasive positive selection. A total of 348 gene families of sufficient size were analyzed, and 33 of these were predicted to contain venom genes. We recovered 151 families containing homologs to previously described venoms, and 40 of these were uniquely gained in Asilidae. Our gene family clustering suggests that many asilidin venom gene families are not natural groupings as originally delimited. Additionally, robber-fly venoms have relatively few sites under positive selection, consistent with the hypothesis that the venom of older lineages are dominated by negative selection acting to maintain toxic function.

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