Genome assemblies are currently being produced at an impressive rate by consortia and individual laboratories. The low costs and increasing efficiency of sequencing technologies now enable assembling genomes at unprecedented quality and contiguity. However, the difficulty in assembling repeat‐rich and GC‐rich regions (genomic “dark matter”) limits insights into the evolution of genome structure and regulatory networks. Here, we compare the efficiency of currently available sequencing technologies (short/linked/long reads and proximity ligation maps) and combinations thereof in assembling genomic dark matter. By adopting different de novo assembly strategies, we compare individual draft assemblies to a curated multiplatform reference assembly and identify the genomic features that cause gaps within each assembly. We show that a multiplatform assembly implementing long‐read, linked‐read and proximity sequencing technologies performs best at recovering transposable elements, multicopy MHC genes, GC‐rich microchromosomes and the repeat‐rich W chromosome. Telomere‐to‐telomere assemblies are not a reality yet for most organisms, but by leveraging technology choice it is now possible to minimize genome assembly gaps for downstream analysis. We provide a roadmap to tailor sequencing projects for optimized completeness of both the coding and noncoding parts of nonmodel genomes.

Building the Tree of Life (ToL) is a major challenge of modern biology, requiring advances in cyberinfrastructure, data collection, theory, and more. Here, we argue that phylogenomics stands to benefit by embracing the many heterogeneous genomic signals emerging from the first decade of large-scale phylogenetic analysis spawned by high-throughput sequencing (HTS). Such signals include those most commonly encountered in phylogenomic datasets, such as incomplete lineage sorting, but also those reticulate processes emerging with greater frequency, such as recombination and introgression. Here we focus specifically on how phylogenetic methods can accommodate the heterogeneity incurred by such population genetic processes; we do not discuss phylogenetic methods that ignore such processes, such as concatenation or supermatrix approaches or supertrees. We suggest that methods of data acquisition and the types of markers used in phylogenomics will remain restricted until a posteriori methods of marker choice are made possible with routine whole-genome sequencing of taxa of interest. We discuss limitations and potential extensions of a model supporting innovation in phylogenomics today, the multispecies coalescent model (MSC). Macroevolutionary models that use phylogenies, such as character mapping, often ignore the heterogeneity on which building phylogenies increasingly rely and suggest that assimilating such heterogeneity is an important goal moving forward. Finally, we argue that an integrative cyberinfrastructure linking all steps of the process of building the ToL, from specimen acquisition in the field to publication and tracking of phylogenomic data, as well as a culture that values contributors at each step, are essential for progress.

Accurate gene tree inference is an important aspect of species tree estimation in a summary-coalescent framework. Yet, in empirical studies, inferred gene trees differ in accuracy due to stochastic variation in phylogenetic signal between targeted loci. Empiricists should, therefore, examine the consistency of species tree inference, while accounting for the observed heterogeneity in gene tree resolution of phylogenomic data sets. Here, we assess the impact of gene tree estimation error on summary-coalescent species tree inference by screening ${\sim}2000$ exonic loci based on gene tree resolution prior to phylogenetic inference. We focus on a phylogenetically challenging radiation of Australian lizards (genus Cryptoblepharus, Scincidae) and explore effects on topology and support. We identify a well-supported topology based on all loci and find that a relatively small number of high-resolution gene trees can be sufficient to converge on the same topology. Adding gene trees with decreasing resolution produced a generally consistent topology, and increased confidence for specific bipartitions that were poorly supported when using a small number of informative loci. This corroborates coalescent-based simulation studies that have highlighted the need for a large number of loci to confidently resolve challenging relationships and refutes the notion that low-resolution gene trees introduce phylogenetic noise. Further, our study also highlights the value of quantifying changes in nodal support across locus subsets of increasing size (but decreasing gene tree resolution). Such detailed analyses can reveal anomalous fluctuations in support at some nodes, suggesting the possibility of model violation. By characterizing the heterogeneity in phylogenetic signal among loci, we can account for uncertainty in gene tree estimation and assess its effect on the consistency of the species tree estimate. We suggest that the evaluation of gene tree resolution should be incorporated in the analysis of empirical phylogenomic data sets. This will ultimately increase our confidence in species tree estimation using summary-coalescent methods and enable us to exploit genomic data for phylogenetic inference. [Coalescence; concatenation; Cryptoblepharus; exon capture; gene tree; phylogenomics; species tree.].

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