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We have submitted:

Genomic data files used for population genomic analyses, including raw and trimmed FASTQ files, BAM alignments and index files, VCF files, pseudogenome reference files, locus alignments, PLINK/ADMIXTURE input and output files, sample filtering files, and shell scripts used for dataset generation and filtering (Genomic_Data).

Environmental and spatial data used for linear modeling, including climatic variables, elevation rasters, vegetation rasters, resistance rasters, drainage basin and river shapefiles, lookup tables, figures, and R scripts used to test relationships between genomic, morphological, geographic, and environmental variables (Linear_Models).

Morphological analysis files, including wing measurement data, wing photographs, figures, and R scripts used to quantify and analyze morphological variation among specimens and populations (Morphology_Analysis).

Paleoclimate ecological niche modeling files, including current CHELSA climate data, Last Glacial Maximum, Last Interglacial, and Mid-Holocene bioclimatic raster layers, model prediction rasters, response curves, and R scripts used to evaluate historical and contemporary habitat suitability (Paleo_Niche_Model).

Principal component analysis and genetic differentiation files, including R scripts and figures used to evaluate population structure and FST-based genetic differentiation among sampling localities (PCA_Fst_Analyses).

Sampling and locality metadata, including locality information and population assignment files used across genomic, morphological, and environmental analyses (Spreadsheets).

Stairway Plot files used to infer historical changes in effective population size, including folded site-frequency spectrum input files, blueprint files, replicate output files, final demographic output files, figures, and program documentation (Stairway_Plot).

Tajima’s D analysis files, including VCF inputs, sliding-window outputs, site-level summaries, bootstrap outputs, statistical test results, figures, and scripts used to evaluate deviations from neutral expectations among populations (Tajima_D_Analysis).

Data repository description, folder summaries, and file organization information for this Dryad submission (READ_ME.md).

A zipped file of the data described above (Supplemental_Information.zip)

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Data Formats

Spreadsheet and metadata files:

• .csv: Comma-separated files used for locality metadata, population assignments, wing measurements, environmental lookup tables, and summary files generated during downstream analyses (Locality_Information.csv, Wing_Measurements.csv, basin_name_lookup.csv, river_name_lookup.csv, vegedata_key.csv).
• .tsv: Tab-separated files used for pseudogenome or locus summary reports (pseudogenome_92loci_min10pct_tiesN.report.tsv).

Text files:

• .txt: Plain-text files containing sample lists, population files, filtering records, ADMIXTURE cross-validation summaries, BAM/FASTQ lists, and program output summaries (Population_File.txt, bad_samples.txt, bam_list.txt, fastq_list.txt, admixture_CV_summary.txt).

R and shell scripts:

• .R: R scripts used for PCA/FST analyses, linear models, morphology analyses, paleoclimate niche modeling, shapefile preparation, and site-frequency spectrum generation (PCA_Fst_Analyses.R, Linear_Models.R, Morphology_Analysis.R, Paleo_ENM.R, shapefile_making.R, sfs_code_create.R).
• .sh: Shell scripts used for consensus sequence generation, sequence length summaries, VCF filtering, and Stairway Plot execution (consensus.sh, length.sh, vcf_filter.sh, eustalacta.blueprint.sh, eustalacta.blueprint.plot.sh).

Cluster job and output files:

• .job: SLURM job scripts submitted to a computing cluster for batch processing (sort.job).
• .out: Standard output files generated by SLURM jobs run on a computing cluster (slurm-9586985_1.out).

Sequence, alignment, and reference genome files:

• .fas/.fasta: FASTA-format sequence files, including locus alignments and pseudogenome reference files used for genomic analyses (L6.fas, L18.fas, pseudogenome_92loci_min10pct_tiesN.fasta).
• .fai: FASTA index files generated for reference sequence files (pseudogenome_92loci_min10pct_tiesN.fasta.fai).
• .amb/.ann/.bwt/.pac/.sa: BWA index files generated for the pseudogenome reference and used for read mapping (pseudogenome_92loci_min10pct_tiesN.fasta.amb, .ann, .bwt, .pac, .sa).

Read and alignment files:

• .fastq.gz: Compressed raw and trimmed sequencing read files used for genomic analyses (P001_WG12_R1.fastq.gz, P001_WG12_R1_trimmed.fastq.gz).
• .bam: Binary alignment files containing mapped sequencing reads (P001_WG12.bam, P025_WA01.bam).
• .bai: BAM index files used for accessing mapped read alignments (P001_WG12.bam.bai).
• .flagstat.txt/.idxstats.txt: Alignment summary statistics generated from BAM files (P001_WG12.flagstat.txt, P001_WG12.idxstats.txt).

Variant and population genomic files:

• .vcf/.vcf.gz: Variant Call Format files containing SNP datasets used for filtering, PCA, FST, ADMIXTURE, Tajima’s D, and demographic analyses (eustalacta_filter.vcf, eustalacta_raw.vcf.gz, eustalacta_tajima.vcf.gz).
• .csi: Index files associated with compressed VCF files (eustalacta_filter.vcf.gz.csi).
• .bed/.bim/.fam: PLINK binary genotype files used for PCA, ADMIXTURE, and population genomic analyses (admixture_input.bed, admixture_input.bim, admixture_input.fam).
• .P/.Q: ADMIXTURE output files containing inferred allele frequencies and individual ancestry proportions across tested K values (admixture_input.fixed.2.P, admixture_input.fixed.2.Q).
• .prune.in/.prune.out: PLINK linkage-disequilibrium pruning output files listing retained and removed variants (eustalacta_ld.prune.in, eustalacta_ld.prune.out).
• .het/.imiss/.lmiss/.nosex: PLINK quality-control output files summarizing heterozygosity, individual missingness, locus missingness, and sample sex-status information where applicable (qc_raw.het, qc_raw.imiss, qc_raw.lmiss, qc_raw.nosex).

Spatial, raster, and ecological niche modeling files:

• .tif: Raster files used for climatic variables, elevation, vegetation, resistance surfaces, drainage basins, and ecological niche model predictions (bio_1.tif, elev_mod.tif, resistance_raster.tif, modern_prediction_cloglog.tif).
• .shp/.shx/.dbf/.prj/.cpg/.qmd: Shapefile components used for drainage basin and river spatial layers (Drainage_Basins.shp, Rivers.shp).

Image, figure, and report files:

• .HEIC: Wing photograph files used for morphological measurement and analysis (IMG_6073.HEIC, IMG_6075.HEIC).
• .svg: Vector graphics generated from ecological niche model response curves (bio_4_response.svg, bio_6_response.svg).
• .html/.json: Quality-control report files generated during read processing (P001_WG12.html, P001_WG12.json).
• .pdf/.docx: Documentation files associated with Stairway Plot analyses (READMEv2.1.pdf, READMEv2.1.docx).

Stairway Plot files:

• .blueprint: Stairway Plot blueprint file specifying demographic model parameters and input settings (eustalacta.blueprint).
• .addTheta: Stairway Plot output files containing theta-adjusted replicate results from demographic analyses (S_eustalacta_ind_filt_folded-1.22_0.67.addTheta).
• Files with no extension: Stairway Plot folded site-frequency spectrum input and replicate output files (S_eustalacta_ind_filt_folded, S_eustalacta_ind_filt_folded-1).

Log files:

• .log: Log files generated by genomic filtering, PLINK, ADMIXTURE, and related command-line analyses (admixture_input.log, admixture_K1.log, eustalacta_ld.log, qc_raw.log).

Additional repository files:

• .md: Markdown files used to describe the repository structure and contents (READ_ME.md).

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Recommended Software for Data Analysis

Analyses in Genomic_Data require standard command-line bioinformatics software for read processing, read mapping, variant filtering, and population genomic file conversion. Recommended software includes SAMtools/BCFtools, BWA, VCFtools, PLINK, ADMIXTURE, and a Unix/Linux shell environment for running included shell scripts.

Analyses in PCA_Fst_Analyses require R and RStudio, along with R packages for population genomic summaries, ordination, data manipulation, and figure generation. These analyses use VCF- and PLINK-derived files generated from the genomic dataset.

Analyses in Tajima_D_Analysis require VCFtools or equivalent software for calculating windowed Tajima’s D from VCF files, along with R and RStudio for summarizing window-level estimates, conducting bootstrap analyses, running statistical tests, and generating figures.

Analyses in Stairway_Plot require Stairway Plot v2.1.3 and Java. Included files contain folded site-frequency spectrum inputs, blueprint files, shell scripts, replicate outputs, final demographic outputs, and figures.

Analyses in Linear_Models require R and RStudio, along with R packages for linear modeling, raster processing, vector spatial data processing, environmental data extraction, and figure generation. These analyses use climatic raster layers, elevation rasters, vegetation rasters, resistance surfaces, drainage basin shapefiles, and river shapefiles.

Analyses in Morphology_Analysis require R and RStudio for analyzing wing measurement data and generating figures. Image-viewing or measurement software may also be used to inspect the included wing photographs such as Fiji (https://imagej.net/software/fiji/downloads).

Analyses in Paleo_Niche_Model require R and RStudio, along with R packages for ecological niche modeling, raster processing, spatial data manipulation, and figure generation. These analyses use current CHELSA climate layers, paleoclimate bioclimatic rasters, ecological niche model prediction rasters, and response-curve outputs.

Spatial raster and shapefile data in Linear_Models and Paleo_Niche_Model can also be viewed and inspected using GIS software such as QGIS or ArcGIS.

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Usage, Compatibility, and Accessibility

This dataset encompasses a wide breadth of file formats due to the span of analyses conducted within this manuscript, including population genomic, morphological, demographic, ecological niche modeling, and landscape/environmental analyses.

The majority of tabular files, scripts, logs, and summary outputs can be viewed using standard text-editing software such as BBEdit (https://www.barebones.com/products/bbedit/index.html). Raster and shapefile data can be viewed using GIS software such as QGIS or ArcGIS. Figures can be viewed using standard image or vector-graphics software. All programs, code, and data used in this repository are publicly available and free to use unless otherwise noted by the software developers.

Researchers are encouraged to use the original data formats, repeat analyses using our data to evaluate reliability and reproducibility, and email the corresponding author with any additional questions or points of confusion.

We are committed to providing support, clarity, and guidance to facilitate the effective use of these data, with the goal of supporting future analyses using this dataset and related phylogeographic, population genomic, and ecological datasets.

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Descriptions

Spreadsheets

Locality_Information.csv

Specimen locality and metadata file used to organize sampling information for Synthemis eustalacta specimens included in downstream genomic, morphological, and environmental analyses.

• QUBIT: DNA concentration estimate for the specimen extraction.
• GEODE CODE: GEODE specimen code, including taxonomic information associated with the sequenced specimen.
• Field_code: Field or collection code assigned to the specimen.
• Order: Taxonomic rank of the specimen at the ordinal level.
• FAMILY: Taxonomic rank of the specimen at the family level.
• GENUS: Taxonomic rank of the specimen at the genus level.
• SPECIES: Taxonomic rank of the specimen at the species level.
• Scientific name: Full scientific name of the specimen.
• date: Collection date as recorded from specimen label data.
• Year: Calendar year of specimen collection.
• latin date: Collection date formatted using roman numeral month notation.
• Sex: Sex of the specimen, when available.
• Age: Life stage or age class of the specimen.
• Preservation: Preservation method of the specimen.
• Country: Country where the specimen was collected.
• State: State or territory where the specimen was collected.
• Park: Park, reserve, or broader named collection area where the specimen was collected.
• Locality: Descriptive locality where the specimen was collected.
• Elevation_ft: Elevation of the collection locality in feet.
• Elevation_m: Elevation of the collection locality in meters.
• Longitude: Longitude coordinate of the collection locality.
• Latitude: Latitude coordinate of the collection locality.

Population_File.txt

Population assignment and sample metadata file used to link individual sequencing samples to BAM files, locality coordinates, population assignments, and GEODE specimen identifiers for population genomic analyses.

• Sample_ID: Sample identifier used for sequencing and downstream genomic analyses.
• BAM_File: BAM alignment file associated with each sample.
• Row_in_Q_File: Plate row assignment for the individual during AHE sequencing.
• Family: Taxonomic rank of the specimen at the family level.
• Genus: Taxonomic rank of the specimen at the genus level.
• Species: Taxonomic rank of the specimen at the species level.
• CODE: Full specimen code containing specimen identifier and taxonomic information.
• putative_species: Putative species assignment used in downstream analyses.
• pop: Population or sampling locality assignment for each specimen.
• Long: Longitude coordinate for the specimen or sampling locality.
• Lat: Latitude coordinate for the specimen or sampling locality.
• GEODE_Code: GEODE specimen identifier associated with each sample.

Genomic_Data

92_loci_genome

Folder containing aligned AHE loci and files used to generate a pseudogenome reference from 92 anchored hybrid enrichment loci. The aligned AHE loci follow approaches used in Goodman et al. 2023, 2026.

• consensus.sh: Shell script containing an embedded Python script used to generate a pseudogenome consensus sequence from the aligned AHE locus files. The script reads all L*.fas alignments, filters columns based on the minimum fraction of taxa with real nucleotide bases, assigns consensus bases by majority rule, assigns N to tied sites, and writes a pseudogenome FASTA file and summary report.
• L.fas*: FASTA-format aligned AHE locus files used as input for pseudogenome construction. Each file represents one aligned locus. Example: L6.fas.
• length.sh: Shell script used to summarize or evaluate sequence/locus lengths during pseudogenome preparation.
• pseudogenome_92loci_min10pct_tiesN.fasta: Consensus pseudogenome FASTA file generated from the 92 aligned AHE loci. Columns were retained based on a minimum taxon-presence threshold, and tied consensus sites were coded as N.
• pseudogenome_92loci_min10pct_tiesN.fasta.amb: BWA index auxiliary file storing information about ambiguous bases in the pseudogenome reference sequence.
• pseudogenome_92loci_min10pct_tiesN.fasta.ann: BWA index annotation file storing reference sequence names, lengths, and related indexing information for the pseudogenome.
• pseudogenome_92loci_min10pct_tiesN.fasta.bwt: BWA Burrows-Wheeler Transform index file used during read alignment to the pseudogenome reference.
• pseudogenome_92loci_min10pct_tiesN.fasta.fai: FASTA index file generated for the pseudogenome reference, storing contig names, sequence lengths, and file offsets for rapid sequence retrieval.
• pseudogenome_92loci_min10pct_tiesN.fasta.pac: BWA packed-sequence file containing the pseudogenome reference sequence in a compressed binary format used during alignment.
• pseudogenome_92loci_min10pct_tiesN.fasta.sa: BWA suffix-array index file used with the Burrows-Wheeler Transform index for efficient read mapping to the pseudogenome.
• pseudogenome_92loci_min10pct_tiesN.report.tsv: Tab-separated report generated by consensus.sh, with one row per AHE locus. Columns include locus name, number of taxa represented in the alignment, minimum number of taxa required to retain an alignment column, original aligned length, number of retained columns, number of dropped columns, and final consensus sequence length.

bam_files

Folder containing BAM alignment files, BAM index files, alignment summary statistics, VCF files, SNP filtering outputs, PLINK files, ADMIXTURE outputs, and scripts used for variant calling, genotype filtering, PCA/ADMIXTURE preparation, Tajima’s D input generation, and downstream population genomic analyses.

• vcf_filter.sh: Shell script used to call and filter SNPs from BAM files mapped to the 92-locus pseudogenome reference. The script uses bcftools mpileup and bcftools call to generate a raw VCF, performs quality-control summaries with PLINK, identifies individuals with high missingness, extracts biallelic SNPs, adds INFO tags for missingness and minor allele frequency, creates separate VCF files for Tajima’s D/Stairway Plot and PCA/ADMIXTURE analyses, performs LD pruning, converts the LD-pruned VCF to PLINK binary format, and runs ADMIXTURE for K = 1–10.
• bam_list.txt: Text file listing BAM files used as input for variant calling in vcf_filter.sh.
• bad_samples.txt: Text file listing individuals removed due to high missingness during SNP filtering. In vcf_filter.sh, individuals with missingness greater than 0.70 are written to this file and excluded from downstream filtered VCFs.
• P001_WG12.bam, P025_WA01.bam, etc.: BAM alignment files containing sequencing reads mapped to the 92-locus pseudogenome reference. Only representative files are listed here; the folder contains BAM files for each sequenced individual.
• P001_WG12.bam.bai, P025_WA01.bam.bai, etc.: BAM index files associated with each BAM alignment file. These files allow rapid access to mapped reads by genomic coordinate.
• P001_WG12.flagstat.txt, P025_WA01.flagstat.txt, etc.: SAMtools flagstat summary files reporting alignment statistics for each BAM file, including total reads, mapped reads, and related mapping-quality summaries.
• P001_WG12.idxstats.txt, P025_WA01.idxstats.txt, etc.: SAMtools idxstats summary files reporting mapped and unmapped reads across pseudogenome contigs for each BAM file.
• eustalacta_raw.vcf.gz: Raw compressed VCF file generated from BAM files using bcftools mpileup and bcftools call.
• eustalacta_raw.biallelic_snps.vcf.gz: Compressed VCF containing only biallelic SNPs extracted from the raw VCF.
• eustalacta_raw.biallelic_snps.tags.vcf.gz: Biallelic SNP VCF with INFO tags added for site-level missingness and minor allele frequency.
• eustalacta_ind_filt.vcf.gz: Individual-filtered VCF generated after removing low-quality individuals listed in bad_samples.txt.
• eustalacta_ind_filt.tags.vcf.gz: Individual-filtered VCF with INFO tags recalculated after sample removal.
• eustalacta_tajima.vcf and eustalacta_tajima.vcf.gz: VCF files filtered for Tajima’s D and Stairway Plot analyses. These files were filtered using missingness and depth thresholds but do not include a minor allele frequency filter, preserving rare variants for neutrality and demographic analyses.
• eustalacta_filter.vcf and eustalacta_filter.vcf.gz: VCF files filtered for PCA and ADMIXTURE analyses. These files include a minor allele frequency filter, missingness filter, and minimum depth filter.
• eustalacta_ldpruned.vcf.gz: LD-pruned VCF file generated from eustalacta_filter.vcf.gz and used for PCA and ADMIXTURE analyses.
• .csi: Index files associated with compressed VCF files. These allow rapid querying of VCF records by genomic coordinate.
• qc_raw.imiss: PLINK individual-missingness summary file generated from the raw VCF.
• qc_raw.lmiss: PLINK locus-missingness summary file generated from the raw VCF.
• qc_raw.het: PLINK heterozygosity summary file generated from the raw VCF.
• qc_raw.log: PLINK log file generated during raw VCF quality-control analyses.
• qc_raw.nosex: PLINK file listing samples with missing or unspecified sex information.
• eustalacta_ld.prune.in: PLINK output file listing SNPs retained after LD pruning.
• eustalacta_ld.prune.out: PLINK output file listing SNPs removed during LD pruning.
• eustalacta_ld.log and eustalacta_ldpruned.log: PLINK log files generated during LD pruning and LD-pruned VCF creation.
• eustalacta_ld.nosex and eustalacta_ldpruned.nosex: PLINK files listing samples with missing or unspecified sex information during LD-pruning steps.
• admixture_input.bed, admixture_input.bim, and admixture_input.fam: PLINK binary genotype files generated from the LD-pruned VCF and used as input for ADMIXTURE.
• admixture_input.fixed.bed, admixture_input.fixed.bim, and admixture_input.fixed.fam: Modified PLINK binary genotype files prepared for ADMIXTURE. Chromosome identifiers in the BIM file were recoded numerically because ADMIXTURE requires numeric chromosome labels.
• admixture_input.fixed.1.P through admixture_input.fixed.10.P: ADMIXTURE output files containing inferred allele frequencies for each tested number of genetic clusters, K = 1–10.
• admixture_input.fixed.1.Q through admixture_input.fixed.10.Q: ADMIXTURE output files containing individual ancestry proportions for each tested number of genetic clusters, K = 1–10.
• admixture_K1.log through admixture_K10.log: ADMIXTURE log files generated for each tested K value.
• admixture_CV_summary.txt: Text file summarizing ADMIXTURE cross-validation error values across K = 1–10.
• admixture_input.log and admixture_input.nosex: PLINK log and sample sex-status files generated during preparation of ADMIXTURE input files.
• out.log: General command-line output log associated with variant filtering or downstream genomic processing.

fastq_files

Folder containing raw and trimmed paired-end FASTQ files, unpaired read files, read-processing quality-control reports, SLURM output files, and the job script used to align trimmed reads to the 92-locus pseudogenome reference.

• sort.job: SLURM array job script used to align trimmed paired-end FASTQ files to the 92-locus pseudogenome reference. The script reads sample names from fastq_list.txt, identifies the corresponding _R1_trimmed.fastq.gz and _R2_trimmed.fastq.gz files, aligns reads with bwa mem, sorts alignments with samtools sort, indexes the resulting BAM file, and generates flagstat and idxstats alignment summaries for each sample.
• fastq_list.txt: Text file listing sample identifiers used by sort.job to select samples for SLURM array-based read alignment.
• P001_WG12_R1.fastq.gz, P025_WA01_R1.fastq.gz, etc.: Compressed raw forward-read FASTQ files for each sequenced individual. Only representative files are listed here; the folder contains one raw R1 FASTQ file for each sample.
• P001_WG12_R2.fastq.gz, P025_WA01_R2.fastq.gz, etc.: Compressed raw reverse-read FASTQ files for each sequenced individual. Only representative files are listed here; the folder contains one raw R2 FASTQ file for each sample.
• P001_WG12_R1_trimmed.fastq.gz, P025_WA01_R1_trimmed.fastq.gz, etc.: Compressed trimmed forward-read FASTQ files used as input for read alignment to the pseudogenome reference.
• P001_WG12_R2_trimmed.fastq.gz, P025_WA01_R2_trimmed.fastq.gz, etc.: Compressed trimmed reverse-read FASTQ files used as input for read alignment to the pseudogenome reference.
• P001_WG12_unpaired_R1.fastq.gz, P025_WA01_unpaired_R1.fastq.gz, etc.: Compressed unpaired forward reads retained after trimming when the corresponding reverse read was discarded.
• P001_WG12_unpaired_R2.fastq.gz, P025_WA01_unpaired_R2.fastq.gz, etc.: Compressed unpaired reverse reads retained after trimming when the corresponding forward read was discarded.
• P001_WG12.html, P025_WA01.html, etc.: HTML quality-control reports generated during read processing for each sample.
• P001_WG12.json, P025_WA01.json, etc.: JSON-format quality-control reports generated during read processing for each sample.
• slurm-9586985_1.out through slurm-9586985_54.out: Standard output files generated by the SLURM array job during read alignment. Each file corresponds to one array task/sample processed by sort.job.

Genomic_Analysis.R

R script used to analyze population genomic structure from an LD-pruned SNP dataset. The script imports VCF and metadata files, performs genetic PCA and SNP clustering, tests population differentiation with ANOVA and AMOVA, calculates global and pairwise FST statistics, and exports summary tables and figures.

Figures

Folder containing summary tables generated from Fst Analyses.

Linear_Models

Folder containing environmental raster layers, elevation data, resistance rasters, resistance shapefiles, figures, and R scripts used to test relationships among genetic differentiation, geography, elevation, environmental variation, and landscape resistance.

Climatic_Variables

Folder containing raster layers used as environmental predictors in linear model and environmental distance analyses.

• bio_1.tif through bio_19.tif: Bioclimatic raster layers used to characterize environmental variation among sampling localities.
• evapotranspiration.tif: Raster layer representing evapotranspiration used as an environmental predictor.
• NDVI.tif: Normalized Difference Vegetation Index raster used as a vegetation/environmental predictor.
• Perc_NonTree.tif: Raster layer representing percent non-tree cover.
• Perc_NonVeg.tif: Raster layer representing percent non-vegetated cover.
• Perc_TreeCov.tif: Raster layer representing percent tree cover.
• vegedata.tif: Vegetation raster used as an environmental predictor in landscape and environmental analyses.

Elevation

Folder containing elevation data used to calculate elevation differences among population sampling localities.

• elev_mod.tif: Elevation raster used to extract population-level elevation values and calculate pairwise elevational distances among populations.

Figures

Folder containing figures and summary outputs generated from the linear model analyses.

Resistance_Rasters

Folder containing rasterized drainage basin, river, vegetation, and resistance-surface files used to calculate least-cost distances among populations.

• basin_name_lookup.csv: Lookup table linking numeric drainage basin raster IDs to drainage basin names.
• river_name_lookup.csv: Lookup table linking numeric river raster IDs to river names and river hierarchy classes.
• vegedata_key.csv: Lookup table describing vegetation raster classes used in resistance-surface construction.
• d8-9s.tif: Raster template used for generating drainage basin and river raster layers.
• Dbasin.tif: Rasterized drainage basin layer generated from drainage basin shapefiles.
• Dbasin_outline.tif: Rasterized drainage basin boundary layer representing watershed divides or basin outlines.
• major.tif: Rasterized major river layer generated from river shapefiles.
• minor.tif: Rasterized minor river layer generated from river shapefiles.
• vegedata.tif: Vegetation raster used in resistance-surface construction.
• resistance_raster.tif: Composite resistance raster generated from major rivers, minor rivers, drainage basin boundaries, and vegetation data. This raster was used to calculate least-cost distances among population sampling localities.

Resistance_Shapefiles

Folder containing vector spatial files and an R script used to generate raster layers for landscape resistance analyses.

• shapefile_making.R: R script used to convert drainage basin and river shapefiles into raster layers. The script reads drainage basin and river shapefiles, creates lookup tables for basin and river IDs, rasterizes drainage basins, separates major and minor rivers, and generates a drainage basin outline raster for resistance analyses.
• Drainage_Basins.shp: Main shapefile geometry file containing drainage basin polygons.
• Drainage_Basins.shx: Shapefile index file associated with the drainage basin shapefile.
• Drainage_Basins.dbf: Attribute table associated with the drainage basin shapefile.
• Drainage_Basins.prj: Projection information file associated with the drainage basin shapefile.
• Drainage_Basins.cpg: Character encoding file associated with the drainage basin shapefile.
• Drainage_Basins.qmd: Metadata file associated with the drainage basin shapefile.
• Rivers.shp: Main shapefile geometry file containing river line features.
• Rivers.shx: Shapefile index file associated with the river shapefile.
• Rivers.dbf: Attribute table associated with the river shapefile.
• Rivers.prj: Projection information file associated with the river shapefile.
• Rivers.cpg: Character encoding file associated with the river shapefile.
• Rivers.qmd: Metadata file associated with the river shapefile.
• d8-9s.tif: Raster template used for spatial alignment when rasterizing drainage basin and river shapefiles.

Linear_Models.R

R script used to perform genetic distance and landscape/environmental association analyses. The script reads the LD-pruned VCF and population metadata, calculates pairwise FST among populations, computes geographic distances, extracts environmental raster values, performs environmental PCA, constructs a composite resistance surface, calculates least-cost distances, fits isolation-by-distance, isolation-by-elevation, and mixed-effects models for environmental/resistance predictors, compares models with AICc and R², and generates figures and summary tables.

PCA_Fst_Analyses

Folder containing R scripts, figures, and summary outputs used to evaluate genetic population structure and genetic differentiation among sampled populations.

PCA_Fst_Analyses.R

R script used to conduct genetic PCA, population-level PCA summaries, clustering, AMOVA-style permutation tests, and FST analyses. The script reads the LD-pruned VCF from Genomic_Data/bam_files/eustalacta_ldpruned.vcf.gz and sample metadata from Spreadsheets/Population_File.txt, converts VCF data into genetic objects for downstream analysis, calculates principal components, identifies SNP-space clusters, summarizes PC scores by population, tests population-level differences in PC axes, estimates population structure using permutation-based distance analyses, calculates global and pairwise FST, and exports FST summary tables.

Figures

Folder containing figures and output tables generated by PCA_Fst_Analyses.R.

Tajima_D_Analysis

Folder containing scripts, per-population sample lists, per-population Tajima’s D output files, log files, figures, and summary outputs used to estimate and visualize Tajima’s D across sampled populations.

tajima_D_Code.sh

Shell script used to run vcftools --TajimaD for each population. The script uses Genomic_Data/bam_files/eustalacta_tajima.vcf.gz as input, reads per-population sample lists from tajimasD/*.txt, skips populations with fewer than two samples, and calculates Tajima’s D in 10,000 bp windows for each eligible population.

Tajima_D.R

R script used to prepare per-population sample lists, read and summarize Tajima’s D output files, filter finite Tajima’s D windows, calculate population-level mean Tajima’s D, perform block bootstrapping, run binomial sign tests, extract elevation values, test associations between Tajima’s D and elevation, and generate final figures. The script reads Genomic_Data/bam_files/eustalacta_tajima.vcf.gz, Spreadsheets/Population_File.txt, and Linear_Models/Elevation/elev_mod.tif, then writes outputs to Tajima_D_Analysis/tajimasD and Tajima_D_Analysis/Figures.

tajimasD

Folder containing per-population sample lists, Tajima’s D output files, and log files generated by vcftools.

• Armidale_Dist.txt, Boyd_River.txt, Cascades_Campground.txt, etc.: Per-population sample list files used with the vcftools --keep option. Each file contains sample IDs assigned to that population.
• Cascades_Campground.Tajima.D, Gloucester_Tops.Tajima.D, Lake_Catalina.Tajima.D, etc.: Per-population Tajima’s D output files generated by VCFtools. These files contain window-level Tajima’s D estimates calculated in 10,000 bp windows.
• Cascades_Campground.log, Gloucester_Tops.log, Lake_Catalina.log, etc.: VCFtools log files generated during per-population Tajima’s D analyses.

Figures

Folder containing figures and output summaries generated by Tajima_D.R.

Stairway_Plot

Folder containing scripts, input files, output files, program documentation, and figures used to infer historical changes in effective population size from the folded site-frequency spectrum.

sfs_code_create.R

R script used to generate a folded site-frequency spectrum from Genomic_Data/bam_files/eustalacta_tajima.vcf.gz. The script reads the VCF, extracts genotypes, calculates allele counts per site, determines the number of sampled chromosomes, folds the site-frequency spectrum because no outgroup-polarized ancestral state was used, and reports callable variant sites for diagnostic purposes.

Stairway_plot.R

R script used to read Stairway Plot summary outputs from replicate runs, combine effective population size trajectories across random seeds, and generate the final demographic history plot. The script reads .summary files from stairway_plot_v2.1.3/S_eustalacta_ind_filt_folded, plots median effective population size with 12.5 % and 87.5 % intervals, marks key temporal reference points, and exports the figure to Stairway_Plot/Figures/p_stairway.svg.

READMEv2.1.docx and READMEv2.1.pdf

Stairway Plot documentation files included with the analysis directory.

Figures

• p_stairway.svg: Final Stairway Plot figure showing inferred historical changes in effective population size through time.

stairway_plot_v2.1.3

Folder containing Stairway Plot input files, shell scripts, replicate outputs, final summaries, documentation, and executable program files.

• eustalacta.blueprint: Stairway Plot blueprint file specifying input settings and demographic model parameters for the folded site-frequency spectrum analysis.
• eustalacta.blueprint.sh: Shell script generated from the Stairway Plot blueprint and used to run the Stairway Plot analysis.
• eustalacta.blueprint.plot.sh: Shell script used to summarize and plot Stairway Plot output files.
• READMEv2.1.docx and READMEv2.1.pdf: Stairway Plot documentation files included with the program directory.

stairway_plot_v2.1.3/S_eustalacta_ind_filt_folded

Folder containing folded site-frequency spectrum inputs, replicate outputs, final output files, and summary plots for the Stairway Plot analysis.

• input/S_eustalacta_ind_filt_folded-1 through input/S_eustalacta_ind_filt_folded-100: Stairway Plot input replicate files generated from the folded site-frequency spectrum.
• final/S_eustalacta_ind_filt_folded-1.22_0.67.addTheta through final/S_eustalacta_ind_filt_folded-100.22_0.67.addTheta: Final theta-adjusted output files generated from the Stairway Plot analysis.
• rand22/S_eustalacta_ind_filt_folded-1.22_0.67.addTheta through rand22/S_eustalacta_ind_filt_folded-100.22_0.67.addTheta: Replicate theta-adjusted output files generated from the random seed 22 analysis.
• rand43/S_eustalacta_ind_filt_folded-1.43_0.67.addTheta through rand43/S_eustalacta_ind_filt_folded-100.43_0.67.addTheta: Replicate theta-adjusted output files generated from the random seed 43 analysis.
• rand65/S_eustalacta_ind_filt_folded-1.65_0.67.addTheta through rand65/S_eustalacta_ind_filt_folded-100.65_0.67.addTheta: Replicate theta-adjusted output files generated from the random seed 65 analysis.
• rand86/S_eustalacta_ind_filt_folded-1.86_0.67.addTheta through rand86/S_eustalacta_ind_filt_folded-100.86_0.67.addTheta: Replicate theta-adjusted output files generated from the random seed 86 analysis.
• S_eustalacta_ind_filt_folded.final.summary: Final Stairway Plot summary file containing median effective population size estimates and confidence intervals through time.
• S_eustalacta_ind_filt_folded.final.summary.pdf and S_eustalacta_ind_filt_folded.final.summary.png: Summary plots generated from the final Stairway Plot output.
• S_eustalacta_ind_filt_folded.rand22.summary, S_eustalacta_ind_filt_folded.rand43.summary, S_eustalacta_ind_filt_folded.rand65.summary, and S_eustalacta_ind_filt_folded.rand86.summary: Stairway Plot summary files generated from replicate analyses using different random seeds.
• S_eustalacta_ind_filt_folded.rand22.summary.pdf/.png, S_eustalacta_ind_filt_folded.rand43.summary.pdf/.png, S_eustalacta_ind_filt_folded.rand65.summary.pdf/.png, and S_eustalacta_ind_filt_folded.rand86.summary.pdf/.png: Summary plots generated for each random-seed replicate analysis.

stairway_plot_v2.1.3/stairway_plot_es

Folder containing Java class files, JAR dependencies, and license files required to run Stairway Plot.

• .class: Compiled Java class files used by Stairway Plot for demographic inference, training/testing, and output plotting.
• .jar: Java archive dependencies required by Stairway Plot.
• _license.txt: License files associated with bundled Stairway Plot dependencies.

Paleo_Niche_Model

Folder containing current and paleoclimate raster layers, ecological niche modeling scripts, response curves, and prediction rasters used to model contemporary and historical habitat suitability for Synthemis eustalacta.

Paleo_ENM.R

R script used to run the ecological niche modeling pipeline. The script downloads and cleans GBIF occurrence records for Synthemis eustalacta, removes duplicate localities, spatially thins occurrence points, loads current CHELSA climate rasters, crops environmental layers to the study region, removes problematic or highly correlated predictors, samples background points, evaluates Maxnet models with ENMeval, selects a final model, generates response curves, creates modern habitat suitability predictions, applies a 10 % training-omission threshold, and projects the selected model onto paleoclimate layers for the Last Interglacial, Last Glacial Maximum, and Mid-Holocene.

CHELSA_cur_V1_2B_r2_5m

Folder containing current CHELSA climate raster data used as environmental predictors for ecological niche modeling.

• 2_5min/readme.txt: Documentation file associated with the current CHELSA climate raster dataset.

Last_Glacial_Maximum_20ka

Folder containing paleoclimate bioclimatic raster layers for the Last Glacial Maximum, approximately 20 ka, used for hindcasting habitat suitability.

• bio_1.tif through bio_19.tif: Last Glacial Maximum bioclimatic raster layers used as paleoenvironmental predictors.

Last_Interglacial_130

Folder containing paleoclimate bioclimatic raster layers for the Last Interglacial, approximately 130 ka, used for hindcasting habitat suitability.

• bio_1.tif through bio_19.tif: Last Interglacial bioclimatic raster layers used as paleoenvironmental predictors.

Mid_Holocene

Folder containing paleoclimate bioclimatic raster layers for the Mid-Holocene used for hindcasting habitat suitability.

• bio_1.tif through bio_19.tif: Mid-Holocene bioclimatic raster layers used as paleoenvironmental predictors.

Predictions

Folder containing response curves and raster predictions generated by Paleo_ENM.R.

• bio_4_response.svg: Response curve for temperature seasonality.
• bio_6_response.svg: Response curve for minimum temperature of the coldest month.
• bio_15_response.svg: Response curve for precipitation seasonality.
• bio_18_response.svg: Response curve for precipitation of the warmest quarter.
• bio_19_response.svg: Response curve for precipitation of the coldest quarter.
• modern_prediction_cloglog.tif: Continuous modern ecological niche model prediction raster on the cloglog scale.
• modern_prediction_10pct_omission.tif: Binary modern prediction raster generated using a 10 % training-omission threshold.
• Last_Glacial_Maximum_20_ka__continuous.tif: Continuous Last Glacial Maximum habitat suitability projection.
• Last_Glacial_Maximum_20_ka__binary_10pct.tif: Binary Last Glacial Maximum projection generated using the 10 % omission threshold.
• Last_Interglacial_130_ka__continuous.tif: Continuous Last Interglacial habitat suitability projection.
• Last_Interglacial_130_ka__binary_10pct.tif: Binary Last Interglacial projection generated using the 10 % omission threshold.
• Mid_Holocene_continuous.tif: Continuous Mid-Holocene habitat suitability projection.
• Mid_Holocene_binary_10pct.tif: Binary Mid-Holocene projection generated using the 10 % omission threshold.

Morphology_Analysis

Folder containing wing measurement data, specimen wing photographs, figures, and R scripts used to quantify and analyze morphological variation among sampled specimens and populations.

Morphology_Analysis.R

R script used to analyze wing morphology data from Wing_Measurements.csv and generate morphology-related figures and statistical summaries. The script reads specimen-level wing measurements, imputes missing numeric trait values, scales wing and leg measurements by thorax length or hind femur length, performs PCA on thorax-scaled morphology traits, generates PCA plots by population and sex, tests trait differences among populations with Kruskal-Wallis tests, tests sex-based trait differences with Wilcoxon tests, runs PERMANOVA on morphology traits, and exports morphology figures to Morphology_Analysis/Figures.

Wing_Measurements.csv

Comma-separated file containing specimen-level morphological measurements used for wing and body-size analyses. The file includes wing traits, thorax length, hind femur length, sex, population assignment, and associated sample identifiers used to evaluate morphological variation among individuals, populations, and sexes.

wing_photos

Folder containing specimen wing photographs used for morphological measurement and documentation.

• P6113143.JPG, P6113144.JPG, P6133145.JPG, etc.: JPG image files of specimen wings used for measurement or visual reference. Only representative files are listed here; the folder contains all wing photographs used in the morphology analysis.

Figures

Folder containing figures and graphical outputs generated from Morphology_Analysis.R.

• wing_pca_plot.svg: PCA figure showing morphology variation among specimens grouped by population.
• sex_pca_plot.svg: PCA figure showing morphology variation among specimens grouped by sex.
• thorax_scaled_trait_violins_by_sex.svg: Violin plot comparing thorax-scaled morphological trait distributions between female and male specimens.