DOI: 10.5061/dryad.cd546
title: Data from: Ancestral gene flow and parallel organellar genome capture result in extreme phylogenomic discord in a lineage of angiosperms

description: While hybridization has recently received a resurgence of attention from
 systematists and evolutionary biologists, there remains a dearth of case
 studies on ancient, diversified hybrid lineages-clades of organisms that
 originated through reticulation. Studies on these groups are valuable in
 that they would speak to the long-term phylogenetic success of lineages
 following gene flow between species. We present a phylogenomic view of
 Heuchera, long known for frequent hybridization, incorporating all three
 independent genomes: targeted nuclear (~400,000 bp), plastid (~160,000
 bp), and mitochondrial (~470,000 bp) data. We analyze these data using
 multiple concatenation and coalescence strategies. The nuclear phylogeny
 is consistent with previous work and with morphology, confidently
 suggesting a monophyletic Heuchera. By contrast, analyses of both
 organellar genomes recover a grossly polyphyletic Heuchera,consisting of
 three primary clades with relationships extensively rearranged within
 these as well. A minority of nuclear loci also exhibit phylogenetic
 discord; yet these topologies remarkably never resemble the pattern of
 organellar loci and largely present low levels of discord inter alia. Two
 independent estimates of the coalescent branch length of the ancestor of
 Heuchera using nuclear data suggest rare or nonexistent incomplete lineage
 sorting with related clades, inconsistent with the observed gross
 polyphyly of organellar genomes (confirmed by simulation of gene trees
 under the coalescent). These observations, in combination with previous
 work, strongly suggest hybridization as the cause of this phylogenetic
 discord.
description: Concatenated nuclear
 alignment, 70 taxanuclearAlignment.phy.reducedConcatenated nuclear tree, unpartitionedNuclearTreeUnpartitioned.treConcatenated nuclear tree, partitioned by geneNuclearTreePartitionedByGene.treConcatenated nuclear tree, partitioned by splice siteNuclearTreePartitionedBySpliceSite.treChloroplast alignment, 70 taxachloroplastAlignment.phy.reducedChloroplast tree, unpartitionedChloroplastOneIRunpartitioned.treChloroplast tree, partitionedChloroplastOneIRpartitioned.treChloroplast alignment of Sanger locisangerAlignment.phy.reducedChloroplast tree of Sanger lociChloroplastSangerTree.treMitochondrial alignment, 66 taxaMitochondrionAlignment66taxa.phyMitochondrial tree, unpartitionedMitochondrionTree66taxaUnpartitioned.treMitochondrial tree, partitionedMitochondrionTree66taxaPartitioned.treMitochondrial tree, upper 50% of taxa in terms of coverageMitochondrionTreeUpper50.treRAxML nuclear partition file, by geneNuclearPartitionByGene.txtRAxML nuclear partition file, by splice siteNuclearPartitionBySpliceSite.txtRAxML chloroplast partition file, coding vs. non-codingChloroplastPartitionCoding-NonCoding.txtRAxML chloroplast partition file, Sanger loci, by geneChloroplastSangerPartitionByGene.txtRAxML mitochondrial partition file, coding vs. non-codingMitochondrionPartitionCoding-NonCoding.txtASTRAL treeASTRALtree.txtSVDquartets treeSVDquartetstree.txtASTRAL optimal gene trees (in one file) and boostraps (separate files in subdirectory)ASTRAL_OptimalGeneTreesandBootstraps.zipBUCKy gene MCMC distributions, with individual gene alignmentsBUCKyGeneDistributions.zipOnline Appendix 1Table of voucher information, collection localities, SRA accession numbers, and coverage values for nuclear, plastid, and mitochondrial contigs, as well as individual low-copy nuclear genes. A translate table for phylogenetic files in the online appendices is also provided.Appendix1_Coverage statistics and voucher information.xlsxOnline Appendix 2Sanger phylogeny of three plastid regions (trnL-F, rpl32-trnL, and rps16-trnK), included in order to increase outgroup sampling (n =15; added samples summarized in Methods). Bootstrap proportions are plotted on all branches of non-zero length; branch coloration follows Fig. 1.Appendix2_SangerChloroplastTree.pdfOnline Appendix 3All 277 analyzed gene trees as inferred in RAxML. The loci are labeled and arranged by descending order of locus length. All clade bootstraps ≥ 50 are plotted on branches.Appendix3_MLgenetrees.pdfOnline Appendix 4All 277 analyzed Bayesian MCM distributions from MrBayes, summarized as 50% majority consensus trees. The loci are labeled and arranged by decreasing length. All clade posterior probabilities ≥ 50 are plotted on branches.Appendix4_Bayesgenetrees.pdfOnline Appendix 5Species-genotype association file in ASTRAL-II format, also used for SVDquartets and MP-EST.Appendix5_ASTRALalleletable.txtOnline Appendix 6ASTRAL-II analysis performed with individual sequences treated as species to assess potential issues with uncertain species limits. Note that these results strongly resemble the concatenation topology and are highly congruent with the ASTRAL-II topology assigning multiple sequences to putative species.Appendix6_ASTRALnoallelemap.pdfOnline Appendix 7Table of results for the φ tests for recombination.Appendix7_RecombinationTests.pdfOnline Appendix 8NeighborNet split network inferred using the concatenated nuclear data, showing conflict among nuclear loci. The currently recognized sections are indicated by colored circles: orange = sect. Holochloa; blue = sect. Heuchera; green = sect. Bracteatae; pink = sect. Rhodoheuchera.Appendix8_NeighborNet.pdfOnline Appendix 9Comparison of coalescent branch length estimates in BUCKy (with individual sequences treated as species, using all 277 loci, subsampled MCMC, and α = 1) and MP-EST (with multiple sequences assigned to putative species). Only internal branches are shown since infraspecific sampling is required to reliably estimate external branch lengths, causing these to misleadingly take on the maximum possible values when there is only one individual per species. The outgroup branch is not to scale.Appendix9_CoalescentBranchLengths.pdfOnline Appendix 10Table of thermocycler programs and PCR primers for PCR and Sanger sequencing.Appendix10_PCRprogramsandprimers.xlsxOnline Appendix 11Summary of taxa included in the analysis of Sanger-sequenced loci, including voucher information and GenBank accession numbers.Appendix11_SangerGenbank and voucherinformation.xlsxOnline Appendix 12Gene trees simulated under the coalescent using the MP-EST tree.Appendix12_MPESTsimulatedGeneTrees1000.txtOnline Appendix 13Summary of bipartitions in the gene trees of online Appendix 12, plotted on the MP-EST species tree estimate.Appendix13_MP_ESTGeneTreeSimulationSummaryPlottedOnSpeciesTree.pdfOnline Appendix 14Gene trees simulated under the coalescent using the BUCKy tree.Appendix14_BUCKysimulatedGeneTrees1000.txtOnline Appendix 15Summary of bipartitions in the gene trees of online Appendix 14 using the plastid tree.Appendix15_BUCKyGeneTreeSimulationSummaryPlottedOnChloroplastTree.pdfOnline Appendix 16Comparison of BUCKy concordance factors for a completely sampled MCMC analysis (last 90% of samples) and a subsampled analysis (last 10% of samples), using the longest 50 low-copy nuclear loci. Branch labels are concordance factors; all factors ≥ 0.1 are in bold. Only internal branches are shown with concordance factors, as in online Appendix 4.Appendix16_BUCKyBurnIn.pdfOnline Appendix 17Comparison of BUCKy concordance factors for different alpha values in the subsampled MCMC analyses. Branch labels are concordance factors; all factors ≥ 0.1 are in bold. External branch concordance factors are not shown; all external branches (those leading to single OTUs) must have 100% concordance since every group containing one OTU must be present in all trees, and hence these numbers may be misleading.Appendix17_BUCKyAlphaValues.pdfOnline Appendix 18Analysis of the low-copy nuclear concatenated dataset with no partitioning scheme.Appendix18_NuclearTree70taxa_scale.pdfOnline Appendix 19Analysis of the low-copy nuclear concatenated dataset with each genetic locus treated as a separate partition.Appendix19_Partitionednuclear-bygene_scale.pdfOnline Appendix 20Concatenated low-copy nuclear phylogeny inferred in RAxML without partitioning, with four rogue taxa pruned as suggested by RogueNaRok. Branch support in sect. Rhodoheuchera (magenta) are dramatically improved with only the position of H. rubescens var. versicolor remaining uncertain. Labeling and coloring of branches follow Fig. 1.Appendix20_NuclearTreeNoRogues_scale.pdfOnline Appendix 21Analysis of the plastid genome dataset with coding and noncoding regions treated as separate partitions and the second copy of the inverted repeat region (IR) deleted. The outgroup branch is not to scale; otherwise, branches are shown proportional to ML branch lengths; branch coloration follows Fig. 1.Appendix21_Partitionedchloroplast_scale.pdfOnline Appendix 22Analysis of the plastid genome dataset with no partitioning scheme and the second copy of the inverted repeat region (IR) deleted. The outgroup branch is not to scale; otherwise, branches are shown proportional to ML branch lengths; branch coloration follows Fig. 1.Appendix22_Unpartitionedchloroplasttree.pdfOnline Appendix 23Analysis of the mitochondrial genome dataset with coding and noncoding regions treated as separate partitions. The outgroup branch is not to scale; otherwise, branches are shown proportional to ML branch lengths; branch coloration follows Fig. 1.Appendix23_Partitionedmitochondrion_scale.pdfOnline Appendix 24Analysis of the mitochondrial genome dataset with no partitioning scheme. The outgroup branch is not to scale; otherwise, branches are shown proportional to ML branch lengths; branch coloration follows Fig. 1.Appendix24_Unpartitionedmitochondrion_scale.pdfOnline Appendix 25Mitochondrial phylogeny including only samples in upper 50th percentile in terms of coverage (unpartitioned). The topology is largely congruent with the tree containing 66 taxa, indicating that the topology is not sensitive to low-coverage samples. The outgroup branch is not to scale; otherwise, branches are shown proportional to ML branch lengths; branch coloration follows Fig. 1.Appendix25_MitochondrionUpper50_scale.pdfOnline Appendix 26Cluster network depicting conflict between plastid and nuclear topologies; blue branches represent reticulation, while black branches show tree-like relationships held in common between the two phylogenies. The topological conflict is so extreme and involves such distantly related taxa that almost no clades are held in common between the two datasets. In the two input trees, all clades with &lt; 70% bootstrap support were collapsed.Appendix26_chloroplastclusternetwork.pdfOnline Appendix 27Tanglegram comparing mitochondrial and concatenated nuclear phylogenies, optimized in Dendroscope to minimize line crossings. Again, the concatenation tree is used to represent the nuclear phylogeny in order to maintain comparability and to show divergent placement of subspecific samples in the mitochondrial tree. In order to minimize spurious disagreement between phylogenies due to estimation error, all clades with &lt; 70% bootstrap support have been collapsed.Appendix27_Tanglegram_mitochondron_nucleus.pdfOnline Appendix 28Proposed introgression events (from Table 2) mapped onto the ASTRAL topology as a visual representation of hypothesized gene flow. Solid red arrows represent proposed hybridization ancestral to a clade; dotted lines represent hybridization among species or populations only; asterisks mark taxa where there was evidence for introgression but the source of the organellar DNA was unresolved.Appendix28_ASTRALintrogressionEvents.pdfOnline Appendix 29The mitochondrial tree (see Fig. 3), pruned to only mitochondrial clade A, and rerooted with Mitella pentandra to match the plastid tree. This figure demonstrates that the mitochondrial tree differs from the plastid tree in ways that cannot be explained simply by subtree misrooting.Appendix29_MitochondrialcladeArerooted.pdfOnline Appendix 30Four mitochondrial regions that were conserved, easily aligned among taxa, and generally were represented by complete sequences in the main alignment (of which were used, respectively: positions 103,060-129,653; 132,480-142,279; 416; 224-428,828; 452,332-467,783).Appendix30_MitochondrialConservedRegions.pdfOnline Appendix 31ASTRAL-II tree, including only those loci for which ϕ-tests could not detect recombination. Methods were otherwise identical to the main ASTRAL-II analysis.Appendix31_ASTRAL_norecombination.pdf