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Abstract BackgroundUniversal single-copy orthologs are the most conserved components of genomes. Although they are routinely used for studying evolutionary histories and assessing new assemblies, current methods do not incorporate information from available genomic data. ResultsHere, we first determine the influence of evolutionary history on universal gene content and find that across 11,098 genomes of plants, fungi, and animals comprising 2606 taxonomic groups, 215 groups significantly vary from their respective lineages in terms of BUSCO (Benchmarking Universal Single Copy Orthologs) completeness. Additionally, 169 groups display an elevated complement of duplicated orthologs, likely from ancestral whole genome duplication events. Secondly, we investigate the extent of taxonomic congruence in broad BUSCO-derived phylogenies. For 275 suitable families out of 543 tested, sites evolving at higher rates produce at most 23.84% more taxonomically concordant, and at least 46.15% less terminally variable phylogenies compared to lower-rate sites. We find that BUSCO concatenated and coalescent trees have comparable accuracy and conclude that higher rate sites from concatenated alignments produce the most congruent and least variable phylogenies. Finally, we show that undetected, yet pervasive BUSCO gene loss events lead to misrepresentations of assembly quality. To overcome this, we filter a Curated set of BUSCOs (CUSCOs) that provide up to 6.99% fewer false positives compared to the standard search and introduce novel methods for comparing assemblies using gene synteny. ConclusionsOverall, we highlight the importance of considering evolutionary histories during assembly evaluations and release the phyca software toolkit that reconstructs consistent phylogenies and offers more precise assembly assessments. 

Abstract The assembly of genomes from pooled samples of genetically heterogenous samples of conspecifics remains challenging. In this study, we show that high‐quality genome assemblies can be produced from samples of multiple wild‐caught individuals. We sequenced DNA extracted from a pooled sample of conspecific herbivorous insects (Hemiptera: Miridae:Tupiocoris notatus) acquired from a greenhouse infestation in Tucson, Arizona (in the range of 30–100 individuals; 0.5 mL tissue by volume) using PacBio highly accurate long reads (HiFi). The initial assembly contained multiple haplotigs (>85% BUSCOs duplicated), but duplicate contigs could be easily purged to reveal a highly complete assembly (95.6% BUSCO, 4.4% duplicated) that is highly contiguous by short‐read assembly standards (N₅₀ = 675 kb; Largest contig = 4.3 Mb). We then used our assembly as the basis for a genome‐guided differential expression study of host plant‐specific transcriptional responses. We found thousands of genes (N = 4982) to be differentially expressed between our new data from individuals feeding onDatura wrightii(Solanaceae) and existing RNA‐seq data fromNicotiana attenuata(Solanaceae)‐fed individuals. We identified many of these genes as previously documented detoxification genes such as glutathione‐S‐transferases, cytochrome P450s, and UDP‐glucosyltransferases. Together our results show that long‐read sequencing of pooled samples can provide a cost‐effective genome assembly option for small insects and can provide insights into the genetic mechanisms underlying interactions between plants and herbivorous pests.