Country-scale COX1 diversity, spatial genetic structure, and mitochondrial demographic history of Aedes aegypti populations in Colombia - Supplementary information

Table S1. Complete metadata for the 717 Aedes aegypti COX1 sequences included in this study, including sample code, GenBank accession number, department, locality, geographic coordinates, collection year, original code, and source. For shared haplotypes represented across multiple locality-year combinations, separate metadata rows were retained for each sampling event. Script S1. R script used to retrieve and curate GenBank metadata for Colombian Aedes aegypti COX1 sequences using the NCBI Entrez utilities through the rentrez package, including accession retrieval, batch downloading of GenBank and FASTA records, metadata extraction, incorporation of curated locality information from Table S1, and expansion of shared haplotypes across multiple sampling events. Script S2. R workflow used during iterative curation of the compiled Aedes aegypti COX1 dataset, including removal of gap characters prior to BLASTN validation against a mitochondrial COX1 reference, retention of BLAST-supported query regions, MAFFT alignment, evaluation of alternative gap thresholds for trimming poorly aligned terminal regions, and final filtering of alignment positions with gaps in ≥45% of sequences. Script S3. R script used to verify coding-frame integrity of the final Aedes aegypti COX1 alignment, including removal of non-ACGT characters, translation under the invertebrate mitochondrial genetic code, detection and consensus-based repair of internal stop codons, and export of the repaired nucleotide alignment. Script S4. Ecological and climatic associations with BSP-inferred effective population size in Colombian Aedes aegypti. This R script evaluates exploratory associations between Bayesian Skyline Plot-derived effective population size (Ne) and ecological-climatic variables across Colombia. The workflow includes processing BSP skyline estimates, interpolating Ne through time, extracting WorldClim precipitation, minimum temperature, and maximum temperature values for 2000–2024 at 2.5 arc-min resolution, and testing annual, monthly, and ecoregion-stratified Spearman correlations. The script also assigns sampling localities IDEAM/IAvH biome categories (2024), evaluates temporal trends in Ne within ecological units, and exports summary tables and plots used to generate the Figure 4 and Supplementary Figure S3. Script S5. Anthropogenic landscape associations with K2P genetic structure in Colombian Ae. aegypti. This R script evaluates the association between COX1 K2P genetic distances and anthropogenic landscape variables derived from GHSL/GHS datasets. The workflow includes extraction of human population density and built-up index values for sampling localities, assignment of population-density classes, year-specific dbRDA models, global dbRDA constrained by human population density, year-stratified dbRDA constrained by built-up index, and export of summary tables and plots used to generate Figure 5.Supporting figuresFigure S1. Geographic and temporal distribution of Colombian Aedes aegypti COX1 sequences included in this study. (a) Map of Colombia showing sampled localities, with bubble size proportional to the number of COX1 sequences per locality. The inset indicates the geographic position of Colombia in South America. (b) Distribution of sequence counts by department, locality, and collection year. Collection years are represented by the color gradient. This figure complements the sampling information summarized in Table 1 and Table S1. Administrative boundaries and country outlines were obtained from Natural Earth via the `rnaturalearth` and `rnaturalearthhires` R packages. Natural Earth data are in the public domain. Data and terms: [https://www.naturalearthdata.com/downloads/](https://www.naturalearthdata.com/downloads/); [https://www.naturalearthdata.com/about/terms-of-use/](https://www.naturalearthdata.com/about/terms-of-use/). Figure S2. Summary of the gap-threshold filtering procedure applied to the compiled Aedes aegypti COX1 alignment. (a) The trade-off curve shows the effect of alternative gap thresholds on retained alignment positions, retained sequences, and mean ungapped sequence length. (b) The critical-gap profile shows the distribution of highly gapped positions across the alignment, indicating that most problematic sites were concentrated at the alignment ends, with fewer isolated internal positions. (c) The histogram summarizes the distribution of ungapped sequence. Figure S3. Bayesian skyline reconstruction of mitochondrial demographic history in Colombian Aedes aegypti. The dashed line represents the mean inferred effective population size through time, and the shaded area represents the 95% highest posterior density interval. Time is expressed as years before present. Estimates represent mitochondrial maternal-lineage history and should not be interpreted as census population size or genome-wide effective population size. Figure S4. Country-scale associations between BSP-inferred effective population size (Ne) and climatic variables in Colombia. (a) Annual and monthly associations between Ne and precipitation. (b) Annual and monthly associations between Ne and minimum temperature. (c) Annual and monthly associations between mean per-population Ne and maximum temperature. Upper panels show relationships between Ne and mean annual climatic variables, whereas lower panels show relationships between interpolated monthly Ne and monthly climatic variables. Shaded areas represent 95% confidence intervals around fitted linear trends.