The use of high-throughput sequencing technologies to produce genome-scale datasets was expected to settle some long-standing controversies across the Tree of Life, particularly in areas where short branches occur at deep timescales. Instead, these datasets have often yielded many well-supported but conflicting topologies, and highly variable gene-tree distributions. A variety of branch-support metrics beyond the nonparametric bootstrap are now available to assess how robust a phylogenetic hypothesis may be, as well as new methods to quantify gene-tree discordance. We applied ten branch support metrics to an ancient group of marine fishes (Teleostei: Pelagiaria) whose interfamilial relationships have proven difficult to resolve due to a rapid accumulation of lineages very early in its history. We analyzed hundreds of loci including published ultraconserved and newly generated exonic data along with their flanking regions to represent all 16 extant families for more than 150 out of 284 valid species in the group. Branch support was typically lower at inter- than intra-familial relationships regardless of the type of marker. Several nodes that were highly supported with bootstrap had very low site and gene-tree concordance, revealing underlying conflict. Combining exons with their flanking regions increased branch lengths in the deep branches of the pelagiarian tree. Despite this conflict, we were able to identify four consistent interfamilial clades, each comprised of two or three families. Our results demonstrate the limitations of employing current metrics of branch support and species-tree estimation when assessing the confidence of ancient evolutionary radiations and emphasize the necessity to embrace alternative measurements to explore phylogenetic uncertainty and discordance in phylogenomic datasets.

These files include:

Supplementary Figures:

Figures S1 to S9

Supplementary Tables:

Tables S1 to S2 in SI_Pelagiaria.docx

Other Supplementary Materials:

Appendix 1: Dataset 1 – Exons (344 taxa & 1,104 markers)

Appendix 2: Dataset 2 – Exons + flanks (293 taxa & 1,104 markers)

Appendix 3: Dataset 3 – Exons (313 taxa & 1,087 markers)

Appendix 4: Dataset 4 – Exons + flanks (281taxa & 1,087 markers)

Appendix 5: Dataset 5 – Exons (204 taxa & 1,087 markers)

Appendix 6: Dataset 6 – Exons + flanks (204 taxa & 1,087 markers)

Appendix 7: List of alternative topologies tested in PhyParts and Quartet Sampling results for each subset

Appendix 8: Summary of the exon dataset properties on a per locus basis

Appendix 9: Summary of the exons + flanks dataset properties on a per locus basis