Data from: Whole-genome analyses resolve the phylogeny of flightless birds (Palaeognathae) in the presence of an empirical anomaly zone

Palaeognathae represent one of the two basal lineages in modern birds, and comprise the volant (flighted) tinamous and the flightless ratites. Resolving palaeognath phylogenetic relationships has historically proved difficult, and short internal branches separating major palaeognath lineages in previous molecular phylogenies suggest that extensive incomplete lineage sorting (ILS) might have accompanied a rapid ancient divergence. Here, we investigate palaeognath relationships using genome-wide data sets of three types of noncoding nuclear markers, together totalling 20,850 loci and over 41 million base pairs of aligned sequence data. We recover a fully resolved topology placing rheas as the sister to kiwi and emu + cassowary that is congruent across marker types for two species tree methods (MP-EST and ASTRAL-II). This topology is corroborated by patterns of insertions for 4,274 CR1 retroelements identified from multi-species whole genome screening, and is robustly supported by phylogenomic subsampling analyses, with MP-EST demonstrating particularly consistent performance across subsampling replicates as compared to ASTRAL. In contrast, analyses of concatenated data supermatrices recover rheas as the sister to all other non-ostrich palaeognaths, an alternative that lacks retroelement support and shows inconsistent behavior under subsampling approaches. While statistically supporting the species tree topology, conflicting patterns of retroelement insertions also occur and imply high amounts of ILS across short successive internal branches, consistent with observed patterns of gene tree heterogeneity. Coalescent simulations indicate that the majority of observed topological incongruence among gene trees is consistent with coalescent variation rather than arising from gene tree estimation error alone, and estimated branch lengths for short successive internodes in the inferred species tree fall within the theoretical range encompassing the anomaly zone. Distributions of empirical gene trees confirm that the most common gene tree topology for each marker type differs from the species tree, signifying the existence of an empirical anomaly zone in palaeognaths.Supplementary data: species codesspecies_codes.txtSupplementary data: sequence alignmentsalignments.tar.gzSupplementary data: phylogenomic subsamplingphylogenomic_subsampling.tar.gzSupplementary data: RAxML bestML gene treesRAxML_bestML_gene_trees.tar.gzSupplementary data: RAxML CNEE bootstraps part1RAxML_CNEE_bootstraps_part1.tar.gzSupplementary data: RAxML CNEE bootstraps part2RAxML_CNEE_bootstraps_part2.tar.gzSupplementary data: RAxML CNEE bootstraps part3RAxML_CNEE_bootstraps_part3.tar.gzSupplementary data: RAxML intron bootstrapsRAxML_intron_bootstraps.tar.gzSupplementary data:RAxML UCE bootstrapsRAxML_UCE_bootstraps.tar.gzSupplementary data: coalescent simulationscoalescent_simulations.tar.gzSupplementary data: CR1 retroelement insertions final datasetCR1_retroelement_insertions_final_dataset.tar.gzSupplementary data: rooted_tripletsrooted_triplets.tar.gzSupplementary data: species_treesspecies_trees.tar.gzSupplementary data: CR1 retroelement insertions bioinformatic pipeline part1CR1_retroelement_insertions_bioinformatic_pipeline_part1.tar.gzSupplementary data: CR1 retroelement insertions bioinformatic pipeline part2CR1_retroelement_insertions_bioinformatic_pipeline_part2.tar.gzSupplementary data: CR1 retroelement insertions bioinformatic pipeline part3CR1_retroelement_insertions_bioinformatic_pipeline_part3.tar.gzSupplementary data: CR1 retroelement insertions bioinformatic pipeline part4CR1_retroelement_insertions_bioinformatic_pipeline_part4.tar.gzSupplementary data: CR1 retroelement insertions bioinformatic pipeline part5CR1_retroelement_insertions_bioinformatic_pipeline_part5.tar.gzSupplementary data: CR1 retroelement insertions bioinformatic pipeline part6CR1_retroelement_insertions_bioinformatic_pipeline_part6.tar.gzSupplementary data: CR1 retroelement insertions bioinformatic pipeline part7CR1_retroelement_insertions_bioinformatic_pipeline_part7.tar.gzSupplementary data: CR1 retroelement insertions bioinformatic pipeline part8CR1_retroelement_insertions_bioinformatic_pipeline_part8.tar.gzSupplementary data: CR1 retroelement insertions bioinformatic pipeline part9CR1_retroelement_insertions_bioinformatic_pipeline_part9.tar.gzSupplementary data: CR1 retroelement insertions bioinformatic pipeline part10CR1_retroelement_insertions_bioinformatic_pipeline_part10.tar.gzSupplementary data: CR1 retroelement insertions bioinformatic pipeline part11CR1_retroelement_insertions_bioinformatic_pipeline_part11.tar.gzSupplementary data: CR1 retroelement insertions bioinformatic pipeline part12CR1_retroelement_insertions_bioinformatic_pipeline_part12.tar.gzSupplementary data: CR1 retroelement insertions bioinformatic pipeline part13CR1_retroelement_insertions_bioinformatic_pipeline_part13.tar.gzSupplementary data: CR1 retroelement insertions bioinformatic pipeline part14CR1_retroelement_insertions_bioinformatic_pipeline_part14.tar.gzSupplementary data: CR1 retroelement insertions bioinformatic pipeline part15CR1_retroelement_insertions_bioinformatic_pipeline_part15.tar.gzSupplementary Figure S1Supplementary Figure S1 Palaeognath phylogenetic relationships inferred with MP-EST (a-d) and ASTRAL (e-h) coalescent-based species tree methods and maximum-likelihood inference of fully partitioned concatenated alignments with ExaML (i-l). Topologies were inferred from 12,676 conserved non-exonic elements (CNEEs; parts a,e,i), 5,016 introns (parts b,f,j), 3,158 ultraconserved elements (UCEs; parts c,g,k), and the total evidence data set of 20,850 loci combined across marker types (TENT; parts d,h,l). Bootstrap supports are drawn for clades with < 100% support.Suppl_FigS1.pdfSupplementary Figure S2Supplementary Figure S2 Support for alternative hypotheses of the sister group to emu + cassowary from phylogenomic subsampling using MP-EST (a-c), ASTRAL (d-f), and ExaML (g-i). Plots display the mean bootstrap support for each hypothesis from 10 replicates of randomly sampled loci within each data set size category (e.g. 50–3000 loci, shown on x-axis).Suppl_FigS2.pdfSupplementary Figure S3Supplementary Figure S3 Heatmap of the number of replicates from phylogenomic subsampling that support each alternative hypothesis for the sister group to emu + cassowary at a minimum bootstrap support of 70% (a-i) and 90% (j-r), with results from each analysis method displayed in separate panels (e.g. MP-EST a-c & j-l, ASTRAL d-f & m-o, ExaML g-I & p-r). Rows labelled H1-H4 within each panel correspond to the four alternative hypotheses outlined in the legend. Columns within each panel labelled 50-3000 indicate the number of loci subsampled at random with replacement from all loci for each marker type. Coloring of cells indicates the number of replicates (of 10 in total for each data set size category) that support each hypothesis at the given bootstrap cut off, corresponding to the colouring scheme outlined in the legend.Suppl_FigS3.pdfSupplementary Figure S4Supplementary Figure S4 Support for the four most common gene tree topologies for each marker type. Median bootstrap support (a-c) and the median number of substitutions under a parsimony criterion (d-f) for branches in gene tree topologies that conflict with the species tree are shown for each marker type. Letters below each group refer to topologies shown in (j), with numbers referring to branches labelled on those topologies. Reference lines in (a-c) are drawn at 50% bootstrap support. (g-i) Violin plots of ΔAIC for loci belonging to each AGT category for CNEEs (part g), introns (part h), and UCEs (part i). Values show the difference in AIC for sequence alignments given the majority rule extended consensus gene tree topology, relative to the AIC when sequence alignments are constrained to the species tree topology. A reference line is drawn at ΔAIC(gene tree–species tree)= -2, with values beneath this cut off indicating substantially stronger support in favor of the gene tree topology (Burnham and Anderson 2002).Suppl_FigS4.pdfSupplementary Figure S5Supplementary Figure S5 Summary measures of aligned sequence data for each marker type (CNEEs, introns, UCEs), showing the total aligned sequence length per locus (a-c), variable alignment columns as a percent of the total alignment length (d-f), and the percent of parsimony informative alignment columns per locus (g-i).Suppl_FigS5.pdfSupplementary Figure S6Supplementary Figure S6 Pairs of short successive internal branches are consistent with expectations for the anomaly zone. Species tree topologies for CNEEs (a), introns (b), and UCEs (c) are shown with internal branch lengths in coalescent units estimated from best maximum likelihood gene trees with ASTRAL. Terminal branch lengths are uninformative and are drawn as a constant value across taxa. Coalescent branch lengths for all pairs of branches (x and y) are given in (d), with a(x) calculated following Equation 4 from Degnan and Rosenberg (2006). Anomalous gene trees are expected when y < a(x). Clades fulfilling this anomaly zone criterion are shaded in (d), with the corresponding branches indicated in a-c.Suppl_FigS6.pdfSupplementary TablesSupplementary_Tables.pdfREADM