The evolutionary histories of species are not measured directly, but estimated using genealogies inferred for particular loci. Individual loci can have discordant histories, but in general we expect to infer evolutionary histories more accurately as more of the genome is sampled. High Throughput Sequencing (HTS) is now providing opportunities to incorporate thousands of loci in 'phylogenomic' studies. Here, we used target enrichment to sequence c.3000 protein-coding exons in a group of Australian skink lizards (crown group age c.80 Ma). This method uses synthetic probes to 'capture' target exons that were identified in the transcriptomes of selected probe design (PD) samples. The target exons are then enriched in sample DNA libraries prior to performing HTS. Our main goal was to study the efficacy of enrichment of targeted loci at different levels of phylogenetic divergence from the PD species. In taxa sharing a common ancestor with PD samples up to c.20 Ma, we detected little reduction in efficacy, measured here as sequencing depth of coverage. However, at around 80 Myr divergence from the PD species, we observed an approximately two-fold reduction in efficacy. A secondary goal was to develop a workflow for analysing exon capture studies of phylogenetically diverse samples, while minimizing potential bias. Our approach assembles each exon in each sample separately, by first recruiting short sequencing reads having homology to the corresponding protein sequence. In sum, custom exon capture provides a complement to existing, more generic target capture methods and is a practical and robust option across low-moderate levels of phylogenetic divergence.

A fundamental challenge in resolving evolutionary relationships across the tree of life is to account for heterogeneity in the evolutionary signal across loci. Studies of marsupial mammals have demonstrated that this heterogeneity can be substantial, leaving considerable uncertainty in the evolutionary timescale and relationships within the group. Using simulations and a new phylogenomic data set comprising nucleotide sequences of 1550 loci from 18 of the 22 extant marsupial families, we demonstrate the power of a method for identifying clusters of loci that support different phylogenetic trees. We find two distinct clusters of loci, each providing an estimate of the species tree that matches previously proposed resolutions of the marsupial phylogeny. We also identify a well-supported placement for the enigmatic marsupial moles (Notoryctes) that contradicts previous molecular estimates but is consistent with morphological evidence. The pattern of gene-tree variation across tree-space is characterized by changes in information content, GC content, substitution-model adequacy, and signatures of purifying selection in the data. In a simulation study, we show that incomplete lineage sorting can explain the division of loci into the two tree-topology clusters, as found in our phylogenomic analysis of marsupials. We also demonstrate the potential benefits of minimizing uncertainty from phylogenetic conflict for molecular dating. Our analyses reveal that Australasian marsupials appeared in the early Paleocene, whereas the diversification of present-day families occurred primarily during the late Eocene and early Oligocene. Our methods provide an intuitive framework for improving the accuracy and precision of phylogenetic inference and molecular dating using genome-scale data.

Phylogeography, and its extensions into comparative phylogeography, have their roots in the layering of gene trees across geography, a paradigm that was greatly facilitated by the nonrecombining, fast evolution provided by animal mtDNA. As phylogeography moves into the era of next-generation sequencing, the specter of reticulation at several levels-within loci and genomes in the form of recombination and across populations and species in the form of introgression-has raised its head with a prominence even greater than glimpsed during the nuclear gene PCR era. Here we explore the theme of reticulation in comparative phylogeography, speciation analysis, and phylogenomics, and ask how the centrality of gene trees has fared in the next-generation era. To frame these issues, we first provide a snapshot of multilocus phylogeographic studies across the Carpentarian Barrier, a prominent biogeographic barrier dividing faunas spanning the monsoon tropics in northern Australia. We find that divergence across this barrier is evident in most species, but is heterogeneous in time and demographic history, often reflecting the taxonomic distinctness of lineages spanning it. We then discuss a variety of forces generating reticulate patterns in phylogeography, including introgression, contact zones, and the potential selection-driven outliers on next-generation molecular markers. We emphasize the continued need for demographic models incorporating reticulation at the level of genomes and populations, and conclude that gene trees, whether explicit or implicit, should continue to play a role in the future of phylogeography.monsoon tropics | introgression | comparative phylogeography | species trees | coalescent theory