DOI: 10.5061/dryad.gmsbcc2vh
title: Data from: macroevolutionary patterns behind a classic case of coevolution: uncovering the evolution of milkweed longhorn beetles Tetraopes, Cerambycidae

description: 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.
description: 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 <em>T.
 tetrophthalmus</em> 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 <em>T.
 tetrophthalmus</em>, 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
 <em>T. tetrophthalmus</em> 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 <em>T. tetraophthalmus</em> 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
 <em>Tetraopes</em> genomes was unknown, we used the genome
 size of <em>Anoplophora glabripennis</em>(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 <em>T.
 tetraophthalmus</em> (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 <em>Tetraopes</em> 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 <em>T. tetrophthalmus</em> (generated
 in this work), <em>Anoplophora glabripennis</em> [28,
 Agla_2.0], <em>Doliops geometrica</em> Waterhouse, 1842 (Van
 Dam, unpublished data), <em>Aprophata</em> aff.
 <em>notha</em>(Newman, 1842) (Van Dam, unpublished data),
 <em>Achriotypa basalis</em> Pascoe, 1875 (NHI Accession
 No. SRR15249232), <em>Similosodus venosus</em> (Pascoe, 1867)
 (NHI Accession No. SRR15249233), <em>Rhytiphora diva</em>
 (Thomson, 1860) (NHI Accession No. SRR15249221), and
 <em>Turanoclytus namaganensis</em> (Heyden, 1885) (NHI
 Accession No. SRR16700842). The species used as a base taxon was
 <em>A. glabripennis</em> because it corresponds to the same
 subfamily as <em>Tetraopes</em>beetles. 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].
description: # Macroevolutionary patterns behind a classic case of coevolution:
 Uncovering the evolution of milkweed longhorn beetles
 (<i>Tetraopes</i>, Cerambycidae)
 [https://doi.org/10.5061/dryad.gmsbcc2vh](https://doi.org/10.5061/dryad.gmsbcc2vh) Here are the genome assemblies and UCE custom probes that are part of the project titled 'Macroevolutionary patterns behind a classic case of coevolution: Uncovering the evolution of milkweed longhorn beetles (*Tetraopes*, Cerambycidae)'. Code is available at: [https://github.com/NayeliGutierrez/2023_MacroevolTetraopes ](https://github.com/NayeliGutierrez/2023_MacroevolTetraopes) ## Description of the data and file structure * The files with the extension '**_scaffolds.fasta**' correspond to the genome assemblies of the beetle species included in the analysis. The first section of the file name indicates the species to which they correspond. 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. (See references in manuscript.) * The file '**lamiinae-v1-master-probe-list-DUPE-SCREENED.fasta'** contains the UCE custom probes: 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 *Tetraopes* beetles. 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]. (See references in manuscript.) ## Code/Software Code is available at: [https://github.com/NayeliGutierrez/2023_MacroevolTetraopes ](https://github.com/NayeliGutierrez/2023_MacroevolTetraopes)