This repository contains data from multiple related studies investigating chromosome evolution across Metazoa using ancestral linkage group (ALG) analyses and phylogenetic methods. This dataset contains pairs of BCnS loci that are stable, unstable, or close in a selection of clades. This is Supplementary Table 16 in the manuscript https://doi.org/10.1101/2024.07.29.605683

• Original DOI: https://doi.org/10.5061/dryad.m905qfvbv

Brief Description

This dataset includes:

1. Topological mixing and irreversibility analysis (SupplementaryTable_16.xlsx): Data on clade-defining locus pairs that demonstrate topological linkage and mixing in animal chromosome evolution
2. Comprehensive phylogenetic analysis workflow (newick_and_timetree_20251118/): Complete analytical pipeline including phylogenetic trees, chromosome evolution analyses, and temporal pattern detection across 4,454 animal genomes
3. BCnS Reciprocal Best Hits database (BCnSSimakov2022_current_rbh_202509/): Orthology data files (5,821 RBH files, 3.6GB) generated by the ODP pipeline, containing reciprocal best hit information for all analyzed species. These files form the basis of downstream analyses of chromosome evolution in this manuscript.

Data Extraction Instructions

This dataset is distributed as compressed archives to comply with Dryad's file limits.

Quick Start

After downloading all files from Dryad, extract the data:

bash extract_data.sh

This will:

1. Verify file integrity (checksums)
2. Extract BCnSSimakov2022_current_rbh_202509/ (5,821 RBH files, 3.6 GB)
3. Extract newick_and_timetree_20251118/ (11,101 workflow files, 4.7 GB)
4. Verify successful extraction

Manual Extraction

If you prefer to extract manually:

# Extract RBH database
tar -xzf BCnSSimakov2022_current_rbh_202509.tar.gz

# Extract phylogenetic workflow
tar -xzf newick_and_timetree_20251118.tar.gz

Verify Checksums (Optional)

md5sum -c checksums.md5

Expected Directory Structure After Extraction

.
├── BCnSSimakov2022_current_rbh_202509/    (5,821 files, 3.6 GB)
├── newick_and_timetree_20251118/          (11,101 files, 4.7 GB)
├── README.md                              (this file)
├── SupplementaryTable_16.xlsx
├── extract_data.sh
└── checksums.md5

---

Part 1: Topological Mixing and Irreversibility in Animal Chromosome Evolution

Description of the data and file structure

Definitions of clade-defining locus pairs

Pairs of loci can define the archetypal genome for a clade, and are topologically linked, if (a) the pairs are unique to this clade - that is these two sequences are only present on the same chromosome within genomes in this clade, (b) the pairs are fixed at a stable distance in this clade, regardless of whether they are close or distant, or (c) the distances between the pairs are very small in genomes in this clade (cis- interactions). It is therefore possible that type 3 pairs (small inter-locus distance, or cis-) pairs are also type 2 pairs (stable inter-locus distance), but not vice versa. Type 3 pairs can generally be considered to reflect widely studied micro-syntenic regions, or constraints that act on very short (cis-) distances. Such regions can be easily identified via comparisons of both chromosome- and non chromosome-scale genomes (14, 36, 90). Type 2 pairs, on the other hand, can only be uncovered by averaging dozens to hundreds of chromosome-scale genomes. Type 2 pairs, therefore, may reflect more distal entanglement (supplementary text). Extensive mixing, leading to entangled pairs of many gene enhancer-promoter regulatory links within a defined genomic region (e.g., a topological domain, can be seen to make the domain 'resistant' to random translocations that would break one or more of such regulatory links.

There are many locus pairs that are characteristically stable (type 2) or close (type 3) in any given clade. For example, in Spiralia, there are 2,398 pairs stable in the clade, and 2,471 especially close pairs. While the test for type 1 pairs is binary, the stringency of our search for type 2 and type 3 pairs (one-sided μ-2σ cutoffs, ≥ 50% of species with this pair, Methods) means that these numbers are conservative estimates of the number of identifiable markers. We note that the most strongly stable pairs (type 2) are also close pairs (type 3), and these novel linkages appear to be rarely lost in genomes within the clade once the loci become linked (fig. S10C and F). This property enables stable pairs to provide for a phylogenetic signal, as their retention could comprise irreversible states due to topological or regulatory features (Figure 1). We find 690 stable pairs shared in deuterostomes and 185 pairs in protostomes, with no overlap between the two sets. Similarly, out of 2398 spiralian stable pairs, 1743 and 610 are retained in mollusks and annelids, respectively. Whereas only 497 are shared between mollusks and annelids.

Files and variables

File: SupplementaryTable_16.xlsx

Description: The file contained in this dataset is Supplementary Table S16, the legend of which is reproduced below.

Variables

• Table S16. Individual pairs that define each clade. Key for columns:
  • Column A (nodename): The name of the clade for which this pair of loci is being considered.
  • Column B (taxid): The NCBI taxid for the clade listed in column A.
  • Column C (ortholog1): One of the orthologs from the BCnS ALGs in the pair.
  • Column D (rbh1): The ALG to which ortholog1 belongs.
  • Column E (ortholog2): The other ortholog from the pair.
  • Column F (rbh2): The ALG to which ortholog2 belongs.
  • Column G (close_in_clade): 1 if the pair is especially close in this clade, defined by having an occupancy_in of >50% and a z-score of less than -2 for mean_in_out_ratio_log_sigma.
  • Column H (distant_in_clade): Same as close_in_clade, but the z-score must be greater than 2 in mean_in_out_ratio_log_sigma.
  • Column I (stable_in_clade): Same as close_in_clade, but the z-score must be less than -2 in the column sd_in_out_ratio_log_sigma.
  • Column J (unstable_in_clade): The z-score of this pair must be greater than 2 in the column sd_in_out_ratio_log_sigma.
  • Column K (unique_to_clade): 1 if this pair only appears in this clade in the dataset, and not outside of the clade.
  • Column L (pair): An index that uniquely describes the pair, also recorded in columns D-F.
  • Column M (notna_in): The number of genomes in this clade that had this pair present on the same chromosome.
  • Column N (notna_out): The number of genomes outside of the clade that had this pair present on the same chromosome.
  • Column O (mean_in): The mean distance between the two loci in this pair in all of the genomes in this clade.
  • Column P (sd_in): The standard deviation of the distances between these loci in all the genomes in the clade.
  • Column Q (mean_out): The mean distance between the two loci in this pair in all of the genomes outside the clade.
  • Column R (sd_out): The standard deviation of the distances between these loci in all the genomes outside the clade.
  • Column S (occupancy_in): The fraction of genomes in this clade in the dataset that have this pair of loci on the same chromosome.
  • Column T (occupancy_out): The fraction of genomes outside this clade that have this pair of loci on the same chromosome.
  • Column U (num_species_in): The number of genomes in this clade in this dataset.
  • Column V (num_species_out): The number of genomes outside of this clade in this dataset.
  • Column W (sd_in_out_ratio): The ratio of standard deviations of distances inside this clade versus outside the clade.
  • Column X (sd_in_out_ratio_log): The log column W, sd_in_out_ratio.
  • Column Y (mean_in_out_ratio): The ratio of the mean distances of the loci in genomes in the clade versus outside the clade.
  • Column Z (mean_in_out_ratio_log): The log of column Y, mean_in_out_ratio.
  • Column AA (sd_in_out_ratio_log_sigma): The z-score of column X when considered against the distribution of column X values for the same clade.
  • Column AB (mean_in_out_ratio_log_sigma): The z-score of column Z when considered against the distribution of column Z values for the same clade.

Code/software

An Excel spreadsheet viewer is required to view this file. Options are Microsoft Excel (paid), Google Sheets (free but account required), Apple Pages (included with Mac OS), or Libre Office (free).

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Part 2: BCnS Reciprocal Best Hits (RBH) Database

Directory: BCnSSimakov2022_current_rbh_202509/

Description

This directory contains the Reciprocal Best Hits (RBH) orthology database generated by the ODP (Oxford Dot Plot) pipeline. These files establish orthologous relationships between genes across all analyzed species and the BCnS (Branchiostoma and Ciona plus nematode and Simakov) ancestral linkage groups (ALGs).

Database Metrics:

• Total RBH files: 5,821
• Total size: 3.6 GB
• File format: Tab-separated values (TSV)
• Reference: ODP software https://github.com/conchoecia/odp

File Structure

File Naming Pattern:

BCnSSimakov2022_[SpeciesName]-[TaxID]-[Accession]_xy_reciprocal_best_hits.plotted.rbh

Examples:

• BCnSSimakov2022_Abaxparallelepipedus-102642-GCA964197645.1_xy_reciprocal_best_hits.plotted.rbh
• BCnSSimakov2022_Zygaenaviciae-287404-GCA964271875.1_xy_reciprocal_best_hits.plotted.rbh

File Contents

Each RBH file contains reciprocal best hit orthology assignments between genes in a species genome and the BCnS ALG reference set. These files are used throughout the phylogenetic analysis workflow to:

1. Identify ancestral linkage group (ALG) membership for genes
2. Track ALG conservation and rearrangements across phylogeny
3. Calculate dispersion and fusion metrics (Step 3)
4. Generate perspective chromosome analyses (Step 4)

Usage in Workflow

These files are referenced in the analysis scripts via the placeholder <RBH_DATABASE_PATH>, which has been sanitized from the original path /lisc/data/scratch/molevo/dts/manifold/BCnSSimakov2022_current_rbh_202509/.

For detailed information about RBH file generation and format, see the ODP documentation: https://github.com/conchoecia/odp

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Part 3: Comprehensive Phylogenetic Analysis of Chromosome Evolution

Directory: newick_and_timetree_20251118/

Overview

This directory contains a complete 9-step analytical workflow for phylogenetic analysis of chromosome evolution across 4,406 animal species. The analyses below focus on clades with 50 or more species. There were 141 clades that met this criterion. The workflow includes the generation of time-calibrated trees, ancestral linkage group (ALG) analyses, perspective chromosome analyses, and Fourier analysis of evolutionary patterns. All HPC paths have been sanitized to placeholders.

Summary Metrics:

• Total file count: ~17,000 files
• Total file size: ~9.7 GB
• Number of species analyzed: 4,406
• Number of analysis steps: 9
• Number of clades analyzed: 141

Finding Files for Your Clade of Interest

Quick Search Tips

To find results for your clade of interest, use file search or grep commands:

# Find all files mentioning your clade
find newick_and_timetree_20251118/ -name "*Lepidoptera*"

# Search for clade name in all files
grep -r "Mammalia" newick_and_timetree_20251118/

Per-Clade Analysis Locations

Detailed per-clade results are in:

newick_and_timetree_20251118/step7_tree_analysis_branchstatsvtime/
  branch_stats_output/per_clade_analyses/

Events Over Time Analysis

Location: step7_tree_analysis_branchstatsvtime/branch_stats_output/per_clade_analyses/

For each clade, per-clade analyses of ALG fusions and dispersals per million years. Files are named as: [CladeName]_[NCBI_TaxID]_[plottype].[ext]

Main analysis files (140 clades):

• *_changes_vs_intensity.pdf - Plots of clade-specific ALG fusion or dispersal rates versus species origination or extinction rates
• *_changes_vs_time.pdf - Clade-specific plots of ALG fusions or ALG dispersals over time
• *_event_count_conservation.pdf - Number of total events observed before and after phylogenetic weighting
• *_phylogenetic_weighting_verification.pdf - Effects of phylogenetic weighting on average rates
• *_phylogenetic_weights_diagnostic.pdf - Distribution of patristic-distance-summed phylogenetic weights for the clade
• *_temporal_heatmap.pdf - Heatmap of phylogenetic weight distribution at million-year time slices
• *_changes_vs_age_weighted.tsv - Weighted time-series data for changes
• *_changes_vs_age_unweighted.tsv - Unweighted time-series data for changes

Fourier Analysis (subdirectory: fourier_analysis/)

Location: step7_tree_analysis_branchstatsvtime/branch_stats_output/per_clade_analyses/fourier_analysis/

Detailed temporal pattern analysis for each clade.

For dispersal events (139 clades):

• [CladeName]_dispersals_padded.pdf - Time-series plot of ALG dispersion events with temporal padding
• [CladeName]_dispersals_padded_chunk_support.tsv - Statistical support for temporal windows (chunks)
• [CladeName]_dispersals_padded_rsims.pdf - Randomization simulation results visualized
• [CladeName]_dispersals_padded_rsims.tsv - Randomization simulation data (for significance testing)

For fusion events (121 clades with sufficient data):

• [CladeName]_fusions_padded.pdf - Time-series plot of ALG fusion events with temporal padding
• [CladeName]_fusions_padded_chunk_support.tsv - Statistical support for temporal windows
• [CladeName]_fusions_padded_rsims.pdf - Randomization simulation results visualized
• [CladeName]_fusions_padded_rsims.tsv - Randomization simulation data

Complete List of 141 Analyzed Clades

Invertebrates:

• Acalyptratae, Acanthomorphata, Annelida, Anthophila, Anthozoa
• Apocrita, Apoditrysia, Apoidea, Arachnida, Arthropoda
• Aschiza, Aculeata, Autobranchia, Bifurcata, Bilateria
• Brachycera, Calyptratae, Chelicerata, Chromadorea, Chrysomeloidea
• Cnidaria
• Coleoptera, Crambidae, Crustacea, Cucujiformia, Culicomorpha
• Cyclorrhapha, Demospongiae, Dicondylia, Diptera, Ditrysia
• Drosophila, Drosophilidae, Drosophilinae, Drosophilini, Ecdysozoa
• Endopterygota, Ennominae, Erebidae, Eremoneura, Euheterodonta
• Gastropoda
• Geometridae, Glossata, Hemiptera, Heteroconchia, Heteroneura
• Hexacorallia, Hexapoda, Hymenoptera, Ichneumonidae, Ichneumonoidea
• Imparidentia, Larentiinae, Lepidoptera, Lophotrochozoa, Mandibulata
• Metazoa, Mollusca, Muscomorpha, Myriazoa, Nematocera
• Nematoda, Neolepidoptera, Neoptera, Noctuidae, Noctuinae
• Noctuoidea, Nymphalidae, Obtectomera, Olethreutinae, Pancrustacea
• Papilionoidea, Paraneoptera, Polyneoptera, Polyphaga, Porifera
• Protostomia, Pterygota, Pyraloidea, Rhabditida, Rhabditidae
• Rhabditina, Rhabditomorpha, Satyrinae, Schizophora, Spiralia
• Syrphidae, Syrphoidea, Tortricidae, Unidentata

Fishes:

• Actinopteri, Actinopterygii, Clupeocephala, Ctenosquamata, Cypriniformes
• Cyprinoidei, Euacanthomorphacea, Eupercaria, Eurypterygia, Euteleosteomorpha
• Neopterygii, Ostariophysi, Osteoglossocephalai, Otomorpha, Otophysi
• Ovalentaria, Perciformes, Percomorphaceae, Teleostei

Tetrapods:

• Amniota, Amphiesmenoptera, Archelosauria, Archosauria, Artiodactyla
• Australaves, Aves, Boreoeutheria, Carnivora, Dipnotetrapodomorpha
• Episquamata, Euarchontoglires, Euteleostomi, Eutheria, Glires
• Laurasiatheria, Mammalia, Neoaves, Neognathae, Passeriformes
• Rodentia, Sauria, Telluraves, Tetrapoda, Theria

Deuterostomes:

• Chordata, Deuterostomia, Gnathostomata, Vertebrata

Two clades' phylogenetic positions were modified from the NCBI taxonomy tree (Myriazoa and Parahoxozoa), and these clades were assigned unique names and NCBI taxids.

Special Custom Analyses:

• Myriazoa_-67 (custom phylogeny with Ctenophora sister to other animals)
• Parahoxozoa_-68 (Parahoxozoa grouping)

Analysis Pipeline Overview

The workflow consists of 9 sequential steps:

1. step1_generate_newick/ - Generate NCBI taxonomy tree
2. step2_download_newick_from_timetree/ - TimeTree divergence time calibration
3. step3_dispersal_characterization/ - ALG dispersion analysis
4. step4_persp_chr/ - Perspective chromosome analysis (chromosome evolution tracking)
5. step5_perspchangeplot/ - Change visualization
6. step6_perspchrom_df_to_tree/ - Statistical tree mapping with simulations
7. step7_tree_analysis_branchstatsvtime/ - Time-series analysis (contains per-clade results)
8. step8_plot_tree_analysis/ - Tree visualizations
9. step9_fourier/ - Fourier analysis of evolutionary patterns
   • Note: Step9 scripts generate per-clade fourier analysis files, but these outputs are placed in the step7 directory structure at:
   • step7_tree_analysis_branchstatsvtime/branch_stats_output/per_clade_analyses/fourier_analysis/

Key Output Files

Phylogenetic trees:

• step2.../20251130Tree.calibrated_tree.nwk - Time-calibrated phylogeny

Per-species results:

• step4.../per_species_ALG_presence_fusions.tsv - ALG presence and fusions per species

Per-clade results:

• step7.../branch_stats_output/per_clade_analyses/fourier_analysis/ - 141 clades analyzed
• step7.../branch_stats_output/major_clades_summary.tsv - Summary across all clades

Visualizations:

• step8.../tree*.pdf - Annotated phylogenetic trees
• step8.../matches_tree_composite_image.png - Composite visualization

Fourier analyses:

• step9.../clade_specific_results.txt - Periodic patterns detected (in step9 directory)
• Per-clade fourier results - Generated by step9 scripts but located in:
  • step7.../branch_stats_output/per_clade_analyses/fourier_analysis/

Important Notes

Path Sanitization

All HPC paths have been replaced with placeholders:

• <GENOME_DB_PATH> - Genome assembly database
• <ODP_SCRIPTS_PATH> - ODP software scripts
• <ODP_INSTALL_PATH> - ODP installation
• <RBH_DATABASE_PATH> - RBH database
• <ALG_RBH_FILE> - ALG RBH file
• <PYTHON_ENV> - Python environment paths
• <ANALYSIS_PATH> - Analysis directory paths
• <HPC_PATH> - General HPC paths

Excluded Files

For space efficiency, excluded:

• .pkl cache files
• .err/.out SLURM logs
• changestring_checkpoints/ directory (thousands of files)

All final results and scripts are included.

Software Requirements

• Python 3.7+
• ODP (Oxford Dot Plot) software
• NumPy, Pandas, Matplotlib, SciPy
• BioPython, DendroPy
• UMAP, scikit-learn

Glossary

• ALG (Ancestral Linkage Group): Chromosome segment linked in ancestral species
• Dispersion: Fragmentation of ALGs across multiple chromosomes
• Perspective chromosome analysis: Phylogenetic method tracking chromosome changes
• RBH (Reciprocal Best Hit): Method for identifying orthologous genes
• Synteny: Conservation of gene order along chromosomes
• Time calibration: Assigning absolute ages to phylogenetic nodes

Contact

• Darrin T. Schultz
• ORCID: https://orcid.org/0000-0003-1190-1122

License

CC0 1.0 Universal (CC0 1.0) - https://creativecommons.org/publicdomain/zero/1.0/

Version History

• v20251118 (February 25, 2026): Phylogenetic analysis workflow
  • 4,406 species, 141 clades analyzed
  • 9-step pipeline with paths sanitized
• Original: DOI 10.5061/dryad.m905qfvbv - Topological mixing dataset

---

Complete Directory Tree and File Descriptions

This section provides a comprehensive listing of all directories and files in the newick_and_timetree_20251118/ workflow.

Directory Structure Overview

newick_and_timetree_20251118/
├── step1_generate_newick/                    (6 files)
├── step2_download_newick_from_timetree/      (9 files)
├── step3_dispersal_characterization/          (2 files + 2 species subdirs)
│   ├── alg_dispersion_plots/                 (2 files)
│   └── odp_pairwise_decay/                   (2 species directories)
│       ├── Branchiostomafloridae-7739-GCF000003815.2/  (4,352 files)
│       └── Pectenmaximus-6579-GCF902652985.1/          (4,352 files)
├── step4_persp_chr/                          (8 files)
├── step5_perspchangeplot/                    (2 files)
├── step6_perspchrom_df_to_tree/              (124 files total)
│   └── simulations/                          (100 files)
├── step7_tree_analysis_branchstatsvtime/      (2,220 files total)
│   └── branch_stats_output/                  (3 subdirectories)
│       ├── custom_clade_analyses/            (2 custom clades)
│       ├── global_diagnostics/               (5 files)
│       └── per_clade_analyses/               (1,120 files)
│           └── fourier_analysis/             (845 files)
├── step8_plot_tree_analysis/                 (14 files)
└── step9_fourier/                            (14 files)

Step-by-Step File Descriptions

Step 1: Generate Newick Tree (step1_generate_newick/)

Purpose: Generate NCBI taxonomy tree for all species

Files:

• config.yaml - Configuration file with genome paths
• gen_tree.sh - Script to generate a phylogenetic tree from the NCBI taxonomy
• ncbi_tree.log - Log file from tree generation
• ncbi_tree.nwk - Newick format phylogenetic tree from NCBI taxonomy
• species_list.txt - List of all 4,406 species analyzed (species name, NCBI TaxID, assembly accession)
• subspecies_to_species_conversions.tsv - Mapping of subspecies to species-level taxonomy

Step 2: Download TimeTree Calibration (step2_download_newick_from_timetree/)

Purpose: Time-calibrate phylogeny using TimeTree database (http://www.timetree.org)

Files:

• 20251130Tree.calibrated_tree.nwk - Main output: Time-calibrated phylogenetic tree in Newick format
• 20251130Tree.divergence_times.txt - Pairwise divergence times between all taxa
• 20251130Tree.edge_information.tsv - Edge-level information (branch lengths, node IDs)
• 20251130Tree.node_ages_for_config.tsv - Node ages formatted for config files
• 20251130Tree.node_information.tsv - Node-level metadata (ages, support values)
• 20251130Tree.tree_report.txt - Summary report of tree calibration
• newick_timetree.nwk - Alternative Newick format output
• runlog.txt - Execution log
• run_newick_to_common_ancestors.sh - Execution script

Step 3: Dispersal Characterization (step3_dispersal_characterization/)

Purpose: Calculate ALG dispersion metrics and pairwise chromosomal decay for reference species

Root Files:

• config.yaml - Configuration file
• run_characterize_dispersion.sh - Execution script to run dispersion characterization of Branchiostoma and Pecten (Figure 2)

Subdirectory: alg_dispersion_plots/

Purpose: Conservation ranking and dispersion analysis across all species

Files (2):

• ALG_conservation_ranking_BCnSSimakov2022.tsv - Conservation ranking of all ALGs across the dataset
• ALG_dispersion_BCnSSimakov2022.pdf - Figure 2: Visualization of ALG dispersion patterns across phylogeny

Subdirectory: odp_pairwise_decay/

Purpose: Detailed pairwise chromosomal synteny decay for two reference species

Contains complete pairwise synteny decay analyses between reference species and all 4,348 other species in the dataset.

Directory: Branchiostomafloridae-7739-GCF000003815.2/ (lancelet, Branchiostoma floridae)

• decay_dataframes/ (4,348 TSV files)
  • Pattern: Branchiostomafloridae-7739-GCF000003815.2_vs_[Species]-[TaxID]-[Accession]_chromosomal_decay.tsv
  • Content: Pairwise synteny decay data between lancelet and each target species
  • Columns: Chromosome pairs, synteny scores, decay metrics
• plot_ALG_dispersion/ (1 PDF file)
  • Branchiostomafloridae-7739-GCF000003815.2_ALG_dispersion_by_conservation.pdf - ALG dispersion by conservation rank for lancelet
• plot_overview_sp_sp/ (1 PDF file)
  • Branchiostomafloridae-7739-GCF000003815.2_decay_plot_vs_divergence_time.pdf - Synteny decay vs. divergence time for all pairwise comparisons

Directory: Pectenmaximus-6579-GCF902652985.1/ (scallop, Pecten maximus)

• decay_dataframes/ (4,348 TSV files)
  • Pattern: Pectenmaximus-6579-GCF902652985.1_vs_[Species]-[TaxID]-[Accession]_chromosomal_decay.tsv
  • Content: Pairwise synteny decay data between the scallop and each target species
  • Columns: Chromosome pairs, synteny scores, decay metrics
• plot_ALG_dispersion/ (1 PDF file)
  • Pectenmaximus-6579-GCF902652985.1_ALG_dispersion_by_conservation.pdf - ALG dispersion by conservation rank for scallop
• plot_overview_sp_sp/ (1 PDF file)
  • Pectenmaximus-6579-GCF902652985.1_decay_plot_vs_divergence_time.pdf - Synteny decay vs. divergence time for all pairwise comparisons

Step 4: Per-species Chromosome Analysis (step4_persp_chr/)

Purpose: Track chromosome evolution across phylogeny (perspective chromosome method)

Files:

• locdf.tsv - Location dataframe with genomic coordinates
• missing_taxa_from_calibrated_tree.txt - Taxa in tree but missing from analysis
• other_ecdysozoa.txt - Non-standard ecdysozoan taxa
• output.log - Execution log
• perspchrom.tsv - Main output: Perspective chromosome assignments for all species
• per_species_ALG_presence_fusions.tsv - Per-species summary of ALG presence and fusion events
• run_persp_chr.sh - Execution script
• unique_changes_summary.tsv - Summary of unique chromosome change events

Step 5: Perspective Change Plot (step5_perspchangeplot/)

Purpose: Visualize the relationship between chromosome number and evolutionary changes

Files:

• chrom_number_vs_changes.pdf - Scatter plot of chromosome number vs. evolutionary change events. Used in Fig. 3.
• run_makeplot.sh - Plotting script

Step 6: Perspective Chromosome to Tree (step6_perspchrom_df_to_tree/)

Purpose: Map chromosome changes onto a phylogenetic tree with statistical simulations. Used in Fig. S1.

Root Files (24 total):

• run_persp2tree.sh - Execution script
• 14 PDF files: simulations_[TaxID]_[CladeName].pdf - Simulation results for major clades:
  • Acoela, Bilateria, Cnidaria, Ctenophora, Demospongiae, Deuterostomia
  • Ecdysozoa, Metazoa, Nematoda, Annelida, Porifera, Protostomia, Spiralia

Subdirectory: simulations/

Purpose: Statistical simulations to test the significance of chromosome change patterns

Files (100):

• 100 files: sim_results_[N]_10.tsv (where N = 0-99)
  • Each file contains 10 simulation replicates testing the null hypothesis of random chromosome changes
  • Used to calculate the statistical significance of observed patterns

Step 7: Tree Analysis - Branch Stats vs Time (step7_tree_analysis_branchstatsvtime/)

Purpose: Analyze chromosome evolution rates over time for all clades (≥50 species)

Root Files (5):

• diagnose_correction.sh - Diagnostic script for phylogenetic corrections
• extinction_intensity.tsv - Species extinction intensity estimates
• run_plotbranchstatstime.sh - Main execution script
• run_plotbranchstatstime_protost_minus_clitellata.sh - Custom analysis for Protostomia minus Clitellata
• run_plotbranchstatstime_verte_minus_teleost.sh - Custom analysis for Vertebrata minus Teleostei

Subdirectory: branch_stats_output/

Root Files:

• major_clades_summary.tsv - Summary statistics for major clades
• modified_edge_list.tsv - Edge list with chromosome change annotations
• modified_node_list.tsv - Node list with age and evolutionary rate data

Subdirectory: global_diagnostics/ (5 PDFs)

Global analyses across all clades:

• ALL_event_count_conservation.pdf - Conservation of event counts after phylogenetic weighting
• ALL_phylogenetic_weighting_verification.pdf - Verification of weighting methodology
• ALL_phylogenetic_weights_by_clade.pdf - Distribution of weights across all clades
• ALL_phylogenetic_weights_diagnostic.pdf - Diagnostic plots for weight calculations
• ALL_temporal_heatmap.pdf - Heatmap of evolutionary activity across all time periods

Subdirectory: custom_clade_analyses/ (2 custom clade directories)

Special analyses for clades with modified phylogenetic positions:

Directory: Protostomia_33317_minus_Clitellata_42113/

• 8 analysis files (same types as per_clade_analyses)
• fourier_analysis/ subdirectory with 6 files (3 dispersals, 3 fusions)

Directory: Vertebrata_7742_minus_Teleostei_32443/

• 8 analysis files (same types as per_clade_analyses)
• fourier_analysis/ subdirectory with 6 files (3 dispersals, 3 fusions)

Subdirectory: per_clade_analyses/ (1,120 files)

File Pattern: [CladeName]_[TaxID]_[analysis_type].[ext]

Per-clade analysis files (8 files × 140 clades = 1,120 files):

1. *_changes_vs_intensity.pdf (140 PDFs)
   • Plots chromosome changes (fusions/dispersals) vs. species diversification/extinction rates
   • Tests the correlation between chromosome evolution and diversification
2. *_changes_vs_time.pdf (140 PDFs)
   • Time series of chromosome change rates (fusions and dispersals per million years)
   • Shows temporal patterns of chromosome evolution
3. *_event_count_conservation.pdf (140 PDFs)
   • Comparison of event counts before and after phylogenetic weighting
   • Validates phylogenetic correction methodology
4. *_phylogenetic_weighting_verification.pdf (140 PDFs)
   • Verification that phylogenetic weighting removes branch length bias
   • Shows average rates across species with different amounts of data
5. *_phylogenetic_weights_diagnostic.pdf (140 PDFs)
   • Distribution of phylogenetic weights across species in the clade
   • Diagnostic to identify species with unusual weighting
6. *_temporal_heatmap.pdf (140 PDFs)
   • Heatmap showing distribution of phylogenetic weight across time slices
   • Identifies time periods with more/less data coverage
7. *_changes_vs_age_weighted.tsv (140 TSV files)
   • Data file: Time-series data with phylogenetic weighting applied
   • Columns: time_mya, fusion_rate, dispersal_rate, num_species, total_weight
8. *_changes_vs_age_unweighted.tsv (140 TSV files)
   • Data file: Raw time-series data without phylogenetic correction
   • Columns: time_mya, fusion_count, dispersal_count, num_branches

Subdirectory: per_clade_analyses/fourier_analysis/ (845 files)

Purpose: Detect periodic patterns in chromosome evolution using Fourier analysis

File Patterns:

Dispersal Analysis (141 clades × 4 files = 564 files):

1. [CladeName]_dispersals_padded.pdf (141 PDFs)
   • Time-series plot of ALG dispersal events with temporal padding
   • Shows data, fitted trend, and detected periodicities
2. [CladeName]_dispersals_padded_chunk_support.tsv (141 TSV files)
   • Statistical support for temporal windows (chunks)
   • Columns: chunk_ID, start_mya, end_mya, p_value, significance
3. [CladeName]_dispersals_padded_rsims.pdf (141 PDFs)
   • Visualization of randomization test results
   • Shows observed vs. expected Fourier power spectrum
4. [CladeName]_dispersals_padded_rsims.tsv (141 TSV files)
   • Randomization simulation data for significance testing
   • Columns: frequency, observed_power, mean_random_power, p_value

Fusion Analysis (121 clades × 4 files = 484 files; fewer clades due to insufficient fusion events):

1. [CladeName]_fusions_padded.pdf (121 PDFs)
   • Time-series plot of ALG fusion events with temporal padding
2. [CladeName]_fusions_padded_chunk_support.tsv (121 TSV files)
   • Statistical support for temporal windows in fusion events
3. [CladeName]_fusions_padded_rsims.pdf (121 PDFs)
   • Randomization test visualization for fusion events
4. [CladeName]_fusions_padded_rsims.tsv (121 TSV files)
   • Randomization simulation data for fusion events

Note: Some clades have insufficient fusion events for reliable Fourier analysis, resulting in 121 clades with fusion analysis vs. 141 with dispersal analysis.

Step 8: Plot Tree Analysis (step8_plot_tree_analysis/)

Purpose: Generate annotated phylogenetic tree visualizations

Files (14):

• run_plotannotatedtree.sh - Plotting script
• tree.pdf - Base phylogenetic tree visualization

Bivariate visualizations (chromosome number changes):

• tree_2d_bivariate.pdf - Bivariate tree with fusion/dispersal rates
• tree_2d_bivariate_balanced_purple.pdf - Color scheme variant
• tree_2d_bivariate_lavender.pdf - Color scheme variant
• tree_2d_bivariate_overlay.pdf - Overlay visualization
• tree_2d_bivariate_softer_purple.pdf - Color scheme variant
• tree_2d_bivariate_true_magenta.pdf - Color scheme variant
• tree_2d_bivariate_violet.pdf - Color scheme variant

Diagnostic visualizations:

• tree_diagnostic_dispersals_overlay.pdf - Dispersal events mapped to the tree
• tree_diagnostic_fusions_overlay.pdf - Fusion events mapped to the tree
• tree_split_side.pdf - Split-view tree visualization

Composite images:

• matches_tree_composite_image.png - Clustered composite of a tree with chromosome evolution patterns
• matches_tree_composite_image_unclustered.png - Unclustered composite

Step 9: Fourier Analysis (step9_fourier/)

Purpose: Control scripts for running Fourier analysis on the HPC cluster

Files (14):

• branches_per_age.pdf - Diagnostic: number of phylogenetic branches per time slice
• clade_specific_results.txt - Summary: Detected periodic patterns for all clades
• count_branches.py - Python script to count branches per time window
• fourier_files.txt - List of input files for Fourier analysis
• support_vs_time_window.pdf - Diagnostic: statistical power vs. time window size
• vertebrate_minus_teleost_time.py - Custom script for Vertebrata minus Teleostei analysis

Execution scripts (generate files in step7/fourier_analysis/):

• run_fourier.sh - Main Fourier analysis script
• run_fourier2.sh - Alternative execution script
• run_fourier_array.sh - Array job script for parallel execution
• submit_fourier_array.sh - SLURM submission script
• run_protostomes_minus_clitellata.sh - Custom analysis for Protostomia minus Clitellata
• run_vertebrates.sh - Vertebrate-specific analysis
• run_vertebrates350.sh - Vertebrate analysis with 350 Myr window
• run_vertebrates_loop.sh - Vertebrate analysis in loop

Note: Step 9 scripts generate per-clade Fourier analysis outputs, but these files are placed in:
step7_tree_analysis_branchstatsvtime/branch_stats_output/per_clade_analyses/fourier_analysis/

File Count Summary

Key Figure References

Main Figures in Paper:

• Figure 2: step3_dispersal_characterization/alg_dispersion_plots/ALG_dispersion_BCnSSimakov2022.pdf
  • ALG dispersion patterns across phylogeny showing conservation ranking

Supplementary Figures:

• Synteny decay: step3_dispersal_characterization/odp_pairwise_decay/[Species]/plot_overview_sp_sp/*.pdf
• Per-clade time series: step7_tree_analysis_branchstatsvtime/branch_stats_output/per_clade_analyses/*_changes_vs_time.pdf
• Fourier analysis: step7_tree_analysis_branchstatsvtime/branch_stats_output/per_clade_analyses/fourier_analysis/*.pdf
• Annotated trees: step8_plot_tree_analysis/tree*.pdf