Phylogeny and divergence time estimation of Io moths and relatives (Lepidoptera: Saturniidae: Automeris)

The saturniid moth genus Automeris includes 145 described species. Their geographic distribution ranges from the eastern half of North America to as far south as Peru. Automeris moths are cryptically colored and their forewings resemble dead leaves, with conspicuously colored, elaborate eyespots hidden on their hindwings. Despite their charismatic nature, the evolutionary history and relationships within Automeris and between closely related genera, remain poorly understood. In this study, we present the most comprehensive phylogeny of Automeris to date, including 80 of the 145 described species. We also incorporate two morphologically similar hemileucine genera, Pseudautomeris and Leucanella, as well as a morphologically distinct genus, Molippa. We obtained DNA data from both dry-pinned and ethanol-stored museum specimens and conducted Anchored Hybrid Enrichment (AHE) sequencing to reconstruct a high-quality dataset for phylogenetic analysis. The resulting phylogeny supports Automeris as a paraphyletic genus, with Leucanella and Pseudautomeris nested within, with the most recent common ancestor dating back to 21 mya. This study lays the foundation for future research on various aspects of Automeris biology, including anti-predator defense mechanisms, ecological adaptations, geographical distribution patterns, and potential drivers of speciation.

Methods
We used the “BOM1” Anchored Hybrid Enrichment probe set, which was developed to target 921 loci across Bombycoidea. We used this probe set because it has been proven effective in capturing sequence data from both dry-pinned and ethanol-preserved specimens. AHE sequences were assembled using an Iterative Baited Assembly (IBA) approach. This involves using the original sequencing probes to identify matching raw reads, which are then assembled into a novel probe.

Sequences were aligned using MAFFT v7.245 and a 50% consensus was generated using FASconCAT-G v1.02  with the ‘-c -c -c’ command to result in one sequence per locus per specimen. To minimize the extent of missing data in the final concatenated dataset, loci that were only obtained from three or fewer species were excluded from the datasets. Loci were concatenated across all species into one supermatrix using FASconCAT-G with the ‘-s’ command.

Phylogenetic inference was conducted on concatenated supermatrices using a maximum likelihood analysis with IQTREE v. 1.5.

Divergence time estimation was performed using BEAST v2.6.7 using a fixed ultrametric tree generated by IQTREE. We added a MultiMonophyleticConstraint prior to increase precision and accuracy.