DOI: 10.5061/dryad.tmpg4f57d
title: Data from: Phylogenomic analysis of target enrichment and transcriptome data uncovers rapid radiation and extensive hybridization in slipper orchid genus Cypripedium L.

description: Background and Aims: Cypripedium is the most widespread and
 morphologically diverse genus of slipper orchids. Despite several
 published phylogenies, the topology and monophyly of its infrageneric taxa
 remained uncertain. Here, we aimed to reconstruct a robust section-level
 phylogeny of Cypripedium and explore its evolutionary history using target
 capture data for the first time. Methods:  We used the
 orchid-specific bait set Orchidaceae963 in combination with transcriptomic
 data to reconstruct the phylogeny of Cypripedium based on 913 nuclear
 loci, covering all 13 sections. Subsequently, we investigated discordance
 among nuclear and chloroplast trees, estimated divergence times and
 ancestral ranges, searched for anomaly zones, polytomies, and
 diversification rate shifts, and identified potential gene (genome)
 duplication and hybridization events. Key Results: All sections
 were recovered as monophyletic, contrary to the two subsections within
 sect. Cypripedium. The two subclades within this section did not
 correspond to its subsections but matched the geographic distribution of
 their species. Additionally, we discovered high levels of discordance in
 the short backbone branches of the genus and within sect. Cypripedium,
 which can be attributed to hybridization events detected based on
 phylogenetic network analyses, and incomplete lineage sorting caused by
 rapid radiation. Our biogeographic analysis suggested a Neotropical origin
 of the genus during the Oligocene (~30 Ma), with a lineage of potentially
 hybrid origin spreading to the Old World in the Early Miocene (~22 Ma).
 The rapid radiation at the backbone likely occurred in Southeast Asia
 around the Middle Miocene Climatic Transition (~15–13 Ma), followed by
 several independent dispersals back to the New World. Moreover, the
 Pliocene-Quaternary glacial cycles may have contributed to further
 speciation and reticulate evolution within Cypripedium.
 Conclusions: Our study provided novel insights into the
 evolutionary history of Cypripedium based on high-throughput molecular
 data, shedding light on the dynamics of its distribution and diversity
 patterns from its origin to the present.
description: # Data from: Phylogenomic Analysis of Target Enrichment and Transcriptome
 Data Uncovers Rapid Radiation and Extensive Hybridization in Slipper
 Orchid Genus Cypripedium L.
 [https://doi.org/10.5061/dryad.tmpg4f57d](https://doi.org/10.5061/dryad.tmpg4f57d) ## Description of the data and file structure #### 1\_nuclear\_analyses: ``` 1_original_nuclear_fasta_files: Unaligned fasta files (*.fna) of nuclear loci 2_raw_nuclear_homologs: Raw nuclear homolog trees. Cleaned alignments with Phyx (*.aln-cln) and ML trees with IQ-TREE 2 without bootstrap support (*.treefile) 3_final_nuclear_homologs: 1_all_taxa: Filtered homolog trees containing all taxa in the original dataset (*.treefile; monophyletic clades and paraphyletic grades of same species masked, spurious tips removed with TreeShrink) 2_reduced_taxa: Filtered homolog trees (*.tm) with taxa C_calceolus_50, C_himalaicum_66, C_calceolus_var_par_54, C_guttatum_64, C_calceolus_51, C_tibeticum_72, C_cordigerum_60, C_tibeticum_73 removed due to low loci coverage and text file containing the taxon IDs of the removed taxa 4_nuclear_MO_orthologs: 1_fasta_to_trees: 1_fasta files_all_taxa: Ortholog fasta files containing all taxa in the original dataset (*.fa) 2_fasta_files_reduced: Ortholog fasta files (*.fa) with taxa C_calceolus_50, C_himalaicum_66, C_calceolus_var_par_54, C_guttatum_64, C_calceolus_51, C_tibeticum_72, C_cordigerum_60, C_tibeticum_73 removed due to low loci coverage and a text file containing the taxon IDs of the removed taxa 3_alignments: Cleaned alignments with Phyx (*.aln-cln) derived from aligning the reduced ortholog fasta files with OMM_MACSE 4_ortholog trees: 1_unrooted_gene_trees: Unrooted (*.treefile) ML ortholog gene trees inferred using IQ-TREE 2 with ultra-fast bootstrap support using the cleaned ortholog alignments 2_rooted_gene_trees: Rooted (with pxrr; *.rr) ML ortholog gene trees and a text file containing the taxon IDs of the outgroup taxa 5_concatenated_alns: Output files from the concatenation of the alignments of all 913 nuclear loci 2_analyses: 01_ASTRAL: Input file containing all 913 nuclear ortholog gene trees (gene_trees_1_1.tre) and output original and FigTree-rooted wASTRAL species trees (*.tre) 02_IQ-TREE: Input clean concatenated alignment (*.fa) and output files from the IQ-TREE phylogenetic reconstruction using the concatenated alignment 03_gene_duplication_mapping: 1_extracted_clades: Extracted clades and text file containing ingroup and outgroup information 2_bootstrap_filter: Output files from the subclade orthogroup tree topology method using the bootstrap filtering approach 3_topology_filter: Output files from the subclade orthogroup tree topology method using the topology filtering approach 04_Phyparts: Output files from the Phyparts piecharts analysis performed using the pxrr rooted ML ortholog gene trees (1_nuclear_analyses/4_nuclear_MO_orthologs/1_fasta_to_trees/4_ortholog trees/2_rooted_gene_trees) and the nuclear ASTRAL species tree (1_nuclear_analyses/4_nuclear_MO_orthologs/2_analyses/01_ASTRAL) 05_Quartet_Sampling: Output files from the Quartet Sampling analysis performed using the clean concatenated alignment (1_nuclear_analyses/4_nuclear_MO_orthologs/1_fasta_to_trees/5_concatenated_alns) and nuclear ASTRAL species tree (1_nuclear_analyses/4_nuclear_MO_orthologs/2_analyses/01_ASTRAL) 06_anomaly_zone: Python script and output text file from the anomaly zone test performed using the nuclear ASTRAL species tree (1_nuclear_analyses/4_nuclear_MO_orthologs/2_analyses/01_ASTRAL) 07_ASTRAL_polytomy_test: Output tree (*.tre) annotated with p-value labels resulting from the ASTRAL polytomy test performed using the the nuclear ASTRAL species tree and a file containing all 913 nuclear ortholog gene trees (1_nuclear_analyses/4_nuclear_MO_orthologs/2_analyses/01_ASTRAL) 08_PhyloNet: 1_backbone: 1_reduced_gene_trees: Gene trees (*.tm) containing only the taxa selected to investigate backbone hybridization events, and a file containing the taxon IDs of the removed taxa 2_input_files: A file (*.tre) containing all the reduced gene trees and a PhyloNet configuration file (*.nex) used to test for one to ten hybridization events at the backbone 3_output_files: All output files (*.txt) from testing for one to ten hybridization events at the backbone, containing the ten best networks each 2_C_x_alaskanum: 1_reduced_gene_trees: Gene trees (*.tm) containing only the taxa selected to investigate hybridization events within the subclade including Cypripedium x alaskanum, and a file containing the taxon IDs of the removed taxa 2_input_files: A file (*.tre) containing all the reduced gene trees and a PhyloNet configuration file (*.nex) used to test for one hybridization event within the subclade 3_output_files: The output file (*.txt) from testing for one hybridization event within the subclade including C. x alaskanum, containing the ten best networks 3_C_x_columbianum: 1_reduced_gene_trees: Gene trees (*.tm) containing only the taxa selected to investigate hybridization events within the subclade including Cypripedium x columbianum, and a file containing the taxon IDs of the removed taxa 2_input_files: A file (*.tre) containing all the reduced gene trees and a PhyloNet configuration file (*.nex) used to test for one to ten hybridization event within the subclade 3_output_files: All output files (*.txt) from testing for one to ten hybridization events within the subclade including C. x columbianum, containing the ten best networks each 4_C_x_ventricosum: 1_reduced_gene_trees: Gene trees (*.tm) containing only the taxa selected to investigate hybridization events within the subclade including Cypripedium x ventricosum, and a file containing the taxon IDs of the removed taxa 2_input_files: A file (*.tre) containing all the reduced gene trees and a PhyloNet configuration file (*.nex) used to test for one to ten hybridization event within the subclade 3_output_files: All output files (*.txt) from testing for one to ten hybridization events within the subclade including C. x ventricosum, containing the ten best networks each 09_BEAST2: 1_SortaDate: 1_SortaDate_analysis: Output files (*.txt) from the SortaDate analysis performed using the pxrr rooted ML ortholog gene trees (1_nuclear_analyses/4_nuclear_MO_orthologs/1_fasta_to_trees/4_ortholog trees/2_rooted_gene_trees) and the concatenation-based nuclear tree (1_nuclear_analyses/4_nuclear_MO_orthologs/2_analyses/02_IQ-TREE) best_genes_aln_files: The clean alignment files (*.aln-cln) of the 20 best genes selected by the SortaDate analysis 2_concatenated_20_best_genes: Output files for the concatenation of the 20 best genes 2_runs: 0_starting_tree: Newick format of wASTRAL used as the starting tree (*.tre) without LPP support labels 1_run_A: Input (*.xml) and output files for the first BEAST2 run (-seed 123456789) 2_run_B: Input (*.xml) and output files for the second BEAST2 run (-seed 2162134795911422075) 3_run_C: Input (*.xml) and output files for the third BEAST2 run (-seed 8503081007213733102) 4_run_D: Input (*.xml) and output files for the fourth BEAST2 run (-seed 4566870218956303049) 5_sample_prior_run: Input (*.xml) and output files for the BEAST2 run sampling from the prior (-seed 281228161096942447) 3_LogCombiner: Output file from LogCombiner (*.trees) 4_TreeAnnotator: Output file from TreeAnnotator (*.nex) 10_BAMM: 1_input_files: 1_input_tree: Input tree (*.newick) created by removing certain taxa listed in an included text file (namely: non-Cypripedium taxa; Cypripedium taxa not accepted by Frosch and Cribb, 2012; taxon duplicates; hybrids; varieties) from the dated phylogeny created with BEAST2 (1_nuclear_analyses/4_nuclear_MO_orthologs/2_analyses/09_BEAST2/4_TreeAnnotator) 2_BAMM_files: A file (*.txt) including the estimated section-specific sampling fractions based on the classification by Frosch and Cribb (2012) and the control file used to perform the BAMM analysis (*.txt) 2_output_files: Output files (*.txt and *.pdf) from the BAMM analysis 11_BioGeoBEARS: 0_input_tree: Input tree (*.newick) created by removing certain taxa listed in two included text files consecutively from the dated phylogeny created with BEAST2 (1_nuclear_analyses/4_nuclear_MO_orthologs/2_analyses/09_BEAST2/4_TreeAnnotator) 1_nine_areas: 1_input_files: The R script (*.R) used to performed the BioGeoBEARS analysis based on nine specified areas and the input text files containing the taxon distribution data and the distance matrix between the nine specified areas in kilometers 2_output_files: Output files (*.txt and *.pdf) from the BioGeoBEARS analysis based on nine specified areas 2_new_vs_old_world: 1_input_files: The R script (*.R) used to performed the BioGeoBEARS analysis based on two specified areas (i.e., the New and the Old World) and the input text file containing the taxon distribution data 2_output_files: Output files (*.txt and *.pdf) from the BioGeoBEARS analysis based on two specified areas ``` ### 2\_chloroplast\_analyses: ``` 1_original_chloroplast_fasta_files: Unaligned fasta files (*.fna) of chloroplast loci 2_alignments: Cleaned alignments with Phyx (*.aln-cln) derived from aligning the fasta files with MACSE 3_concatenated_alns: Output files from the concatenation of the alignments of all 80 chloroplast loci 4_IQ-TREE: Input clean concatenated alignment (*.phy), input model file (*.model), shell executable file (*.sh) and output files for the IQ-TREE phylogenetic reconstruction using the concatenated alignment, including unrooted (*.treefile) and rooted with pxrr (*.treefile.rr) phylogenies 5_Quartet_Sampling: Shell executable file (*.sh) and output files from the Quartet Sampling analysis performed using the clean concatenated alignment (2_chloroplast_analyses/3_concatenated_alns) and chloroplast IQ-TREE phylogeny (2_chloroplast_analyses/4_IQ-TREE) ``` ### 3\_1\_Ks\_plots: ``` 1_Ks_values: 1_within_species: 1_wgd_dmd: Output files (*.tsv) of wgd dmd delineation of whole paranomes within single species 2_wgd_ksd: Output files (*.tsv) of wgd ksd construction of KS age distributions within single species 2_between_species: 1_wgd_dmd: Output files (*.tsv) of wgd dmd delineation of reciprocal best hits (RBHs) between each corresponding species pair 2_wgd_ksd: Output files (*.tsv) of wgd ksd construction of KS age distributions between each corresponding species pair. (edited) ``` ### 4\_Assembly\_references: Extended set of references from orchid genomes and transcriptomes (*.fa) available on the Sequence Read Archive (SRA) of NCBI used to improve gene extractions. ### 5\_Main\_Figures: The published figures might look weird since they were modified during the typesetting. Here, we provide the figures in SVG format. If you have any questions about the data, please do not hesitate to contact Diego F. Morales-Briones at [dfmoralesb@gmail.com](mailto:dfmoralesb@gmail.com) ## Change Log **April 2025**: Added folder `5_Main_Figures`.