Tetraopes beetles are known for their resistance to milkweed plant toxins and their coevolutionary dynamics with milkweed plants (Asclepias). This association is considered a textbook example of coevolution, in which each species of Tetraopes is specialized to feed on one species of Asclepias. A major challenge to investigating coevolution and molecular ecology studies lies in the limited understanding of the evolutionary history of Tetraopes. By integrating genomic, morphological, paleontological, and geographical data, we present a robust phylogeny of Tetraopes and their relatives, using three inference methods with varying subsets of data, encompassing 2 to 12 thousand UCE loci. We elucidate the diversification patterns of Tetraopes species across major biogeographical regions and their colonization of the Americas. Our findings suggest that the genus originated in Central America approximately 21 million years ago during the Miocene and diversified from the Mid-Miocene to the Pleistocene, coinciding with intense geological activity in Central America. Our data suggest that a common ancestor of Tetraopini migrated into North America, likely facilitated by North Atlantic land bridges during the Paleocene. Establishing a densely sampled phylogeny of Tetraopes beetles provides a foundation for investigating micro- and macroevolutionary phenomena, including coevolution and detoxification mechanisms in this ecologically important group.

DNA extraction and library preparation

DNA was extracted from body tissue and legs; legs were punctured to facilitate the action of proteinase-k. MagAttract HMW DNA Kit was used to isolate high molecular weight genomic DNA from T. tetrophthalmus following the manufacturer’s protocol for solid frozen tissue and recommendations from 10x Chromium DNA Extraction from Single Insects [25]. OmniPrep DNA Extraction kit, DNeasy Blood & Tissue Kit, and Qlamp mini kit were used for DNA extractions of other specimens. DNA fragment size was quantified using Qbit 2.0 fluorometric quantification (Invitrogen, USA), a Bioanalyzer, and 0.5% agarose gel electrophoresis. In some cases, DNA was sheared before library preparation in a Covaris M220 (Covaris Inc., USA).

After DNA extraction, samples were divided into two groups for library preparation and sequencing. The first included 30 specimens for which library preparation was performed at the Center for Comparative Genomics of the California Academy of Sciences. Library preparation for these samples was conducted using NEBNext® Ultra™II DNA Library Preparation kit (New England Biolabs Inc, USA) following the manufacturer’s protocol (size selection protocol for fresh samples, and without size-selection for degraded DNA extracted from museum samples) and later sequenced in Illumina Novaseq, 150 bp paired-end reads. The second group included six samples for which an external company performed library preparation. A 10x Genomics Chromium linked-read library was prepared for T. tetrophthalmus, whereas standard Illumina libraries were generated for the other samples. Libraries were sequenced on Illumina HiSequ, 2 x 250 base-pairs (bp), and generated linked reads for T. tetrophthalmus and short paired-end Illumina reads for the other species (S2 Table). Reads were subject to quality control on Fastp 0.23.2 [26]. Illumina universal adapters were removed, and unpaired reads stored for future use in the assembly process.

Genome assembly

The genome of T. tetraophthalmus was assembled in Supernova 2.1 [27], the software designed for Chromium-prepared libraries. Supernova’s guidelines suggest aiming for 56x coverage, which can be calculated using genome size. As the size of Tetraopes genomes was unknown, we used the genome size of Anoplophora glabripennis(Motschulsky, 1854), the only longhorn beetle genome available so far, as an estimate [981 Mb, 28] to compute the number of reads needed for 56x coverage. The result was higher than the number of reads yielded for T. tetraophthalmus (284,563,427), so Supernova was set to use all available reads (maxreads=all). The output was exported in the four fasta styles offered by the software (raw, megabubbles, pseudohap, and pseudohap2), which differ in how microbubble arms and gaps are represented. All other genomes were assembled using SPAdes 3.15, an iterative short-read genome assembly module [29–31]. Paired and unpaired reads were used as an input with k-mer values of 21, 33, 55, 77, 99, and 127, as recommended for read lengths of 150 bp.

Ultraconserved Element (UCE) custom probe set design

An in-silico test of the Coleoptera UCE probe set was performed on six Tetraopes genomes with the PHYLUCE tutorial III [32, 33]. The probe set captured only 380-390 (~33%) of the 1,172 UCEs in the Coleoptera set. However, as 17 museum specimens were to be included in the sampling (some of them over 70 years old), there was the possibility of the number of UCEs captured to be even lower because fragmented DNA decreases the performance of the probes [34, 35].

As UCE probe sets customized for a focal group have resulted in a larger number of recovered loci and improved phylogenomic analysis in other insect groups [36, 37], we designed a customized set of probes for Lamiinae using the PHYLUCE 1.7.1 pipeline [33]. Eight species with high genome quality available were selected to be included in the design (7 Lamiinae and one Cerambycinae), including T. tetrophthalmus (generated in this work), Anoplophora glabripennis [28, Agla_2.0], Doliops geometrica Waterhouse, 1842 (Van Dam, unpublished data), Aprophata aff. notha(Newman, 1842) (Van Dam, unpublished data), Achriotypa basalis Pascoe, 1875 (NHI Accession No. SRR15249232), Similosodus venosus (Pascoe, 1867) (NHI Accession No. SRR15249233), Rhytiphora diva (Thomson, 1860) (NHI Accession No. SRR15249221), and Turanoclytus namaganensis (Heyden, 1885) (NHI Accession No. SRR16700842). The species used as a base taxon was A. glabripennis because it corresponds to the same subfamily as Tetraopesbeetles. At the time of the study, it was the most complete genome available for the group. The other species belong to four Lamiinae tribes spanning the phylogenetic diversity of the subfamily Lamiinae (Apomecynini, Lamiini, Tetraopini, and Pteropliini) [24]. Soft masked files were used following guidelines of previous probe design studies [36, 38].