DOI: 10.5061/dryad.3xsj3txrn
title: Data from: Rapid colonisation of synanthropic stone martens in a highly urbanised region: Insights from temporal and spatial analysis

description: Medium-sized carnivores, including the synanthropic stone marten (Martes
 foina Erxleben, 1777), have shown remarkable adaptability to urbanized and
 fragmented landscapes, facilitating their spread across mainland Europe.
 This study investigates the recolonisation of a highly urbanised region by
 stone martens within two decades, examining spatial and temporal
 genome-wide data (using genotyping by sequencing) to reveal colonisation
 dynamics, sources, and barriers influencing their expansion. Using
 genotypes from 5,536 SNPs across 376 stone martens collected between 1995
 and 2013, our findings indicate that stone martens successfully expanded
 through urban environments, yet dispersal was neither entirely random nor
 strictly distance-dependent. The initial southeastern stronghold (E1)
 showed the lowest genetic diversity and limited spatial expansion, while
 other population sources contributed to recolonisation, highlighting a
 complex, multi-source expansion. Gene flow in the early stages was largely
 confined to E1, progressing northward and eventually enabling exchange
 with a second eastern lineage (E2). Meanwhile, the western lineage
 displayed higher connectivity, occasionally crossing barriers like
 motorways. Motorways, however, significantly shaped recolonization
 patterns, reducing gene flow, while other elements such as built-up areas,
 secondary roads or waterways showed an additional though very small
 effect. Over the study period, genetic patch size increased, indicating
 longer dispersal distances. Gene flow strengthened within both eastern (E1
 and E2) and western populations. Still, the western population diverged in
 two subclusters (W1 and W2) of which one became more differentiated. This
 suggests limited genetic homogenization in the near future. This study
 provides insights into the genetic and ecological dynamics of carnivore
 recolonization in highly fragmented landscapes.
description: Traffic casualties of stone martens were collected from 1995 to
 2013 in Flanders, the northern region of Belgium. A few samples were from
 the Brussels Capital region and one from the Ardennes in the Walloon
 region. DNA from 386 individuals, extracted using the DNeasy Blood
 & Tissue Kit (Qiagen), was used for Genotyping-by-sequencing
 (GBS). A PstI GBS library of all samples, 83 replicated samples with one
 to three replicates each (18 replicates starting from tissue), and 5
 blanks was prepared according to Elshire et al. (2011), and the resulting
 library was sequenced on an Illumina HiSeq 2000 (Cornell University
 Genomics Core Laboratory). Trimming of Illumina
 adaptors and filtering of reads with a minimal length of 50 bp were
 performed using Cutadapt 4.2. Stacks v2.53 process_radtags was used to
 demultiplex raw reads and remove low-quality reads, followed by the
 removal of the restriction enzyme cut site with Cutadapt. We checked read
 quality using FastQC v0.11.9 after each processing step and using MultiQC
 v1.14 after the final step. The software BWA v0.7.17 and more specifically
 the BWA-MEM algorithm was used for the alignment of the reads to the stone
 marten reference genome, the chromosome-length (2n =38) assembly available
 from the DNA Zoo Consortium
 (https://www.dnazoo.org/assemblies/Martes_foina, accessed on 8 September
 2022; Dudchenko et al., 2017, 2018). After alignment, SAM files were
 converted to BAM files and sorted by position order using SAMtools
 v1.16.1. Stacks was used to call SNPs with a reference
 genome (gstacks), allowing a maximum soft-clipping level of 10% of the
 read length. The dataset was converted to VCF format for further analysis
 in RStudio v2022.12.0+353 with R v4.2.0. Initially, we removed variants
 with a read depth of less than 3, a missingness of more than 30%, a minor
 allele frequency (MAF) of less than 0.01 or a minor allele count (MAC) of
 less than 3 using the R package vcfR v1.13.0. Paralogs were identified
 with the R script HDplot developed by McKinney et al. (2017) and removed.
 We further omitted potential paralogs or multicopy loci by removing
 variants with a read depth in the top 5% (i.e., with a read depth &gt;
 50). Next, samples with high missingness (≥ 80%) were removed.
 Further filtering steps were performed using the  R package snpR
 v1.2.5. Filtering thresholds were set to MAF &gt; 0.01, a maximum
 observed heterozygosity of 0.55, and a missingness rate of less than 30%
 per locus. These filtering steps were repeated until all thresholds were
 reached. Subsequently, the same thresholds were applied, but with a
 missingness threshold per sample (&lt; 50%), instead of per locus.
 SNPs were finally pruned for high levels of pairwise linkage
 disequilibrium (LD) when MAF &gt; 0.01, missingness per sample
 &lt; 30%, and a correlation coefficient existed of at least 0.5
 between SNPs within a sliding window of a maximum 50 SNPs, using the R
 package SNPRelate 1.28.0. In order to filter SNPs that
 deviated from Hardy-Weinberg equilibrium (HWE), we first defined genetic
 clusters using principal component analysis (PCA) with the R package
 adegenet v2.1.10, with missing data imputed as the mean value per SNP. We
 grouped samples according to the PCA results, which showed five major
 genetic clusters. Using the R package pegas v1.2, we tested which SNPs
 deviated from HWE (P &lt; 0.05). Those SNPs that tested positive in
 all five clusters were removed. SNPs potentially under selection were
 detected with pcadapt v4.3.3 using five principal components and an FDR
 correction for multiple testing. The method implemented in the R package
 OutFLANK v0.2  was used as a second approach with groups clustered in the
 same five genetic clusters (see earlier). File conversions were performed
 with the R package radiator 1.2.2. One sample failed to
 sequence. After alignment, skipping primary alignments with insufficient
 mapping qualities (1.2%) and excessively soft-clipped primary alignments
 (2.8%), 1493681 loci could be built, which held 144179 variant sites.
 After the first filtering steps using vcfR and HDplot, 5592 SNPs in 378
 samples were retained. A further 56 SNPs were removed that showed
 deviations from HWE in all five genetic clusters. We found only four
 outliers using PC-Adapt, though no outliers with OutFLANK. Therefore, all
 SNPs were retained for further analysis. Two samples had uncertain
 metadata (location and year) and were, therefore, removed. In our final
 set, average missingness per SNP and sample was 13.63%, and consisted of
 376 samples and 5536 SNPs. References:
 Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler
 ES, et al. (2011) A Robust, Simple Genotyping-by-Sequencing (GBS) Approach
 for High Diversity Species. PLoS ONE 6(5): e19379. https://doi.org/10.1371/journal.pone.0019379 Dudchenko, O., Batra, S. S., Omer, A. D., Nyquist, S. K., Hoeger, M., Durand, N. C., Shamim, M. S., Machol, I., Lander, E. S., Aiden, A. P., & Aiden, E. L. (2017). De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds. Science. https://doi.org/aal3327 The Juicebox Assembly Tools module facilitates de novo assembly of mammalian genomes with chromosome-length scaffolds for under $1000<br>Olga Dudchenko, Muhammad S. Shamim, Sanjit S. Batra, Neva C. Durand, Nathaniel T. Musial, Ragib Mostofa, Melanie Pham, Brian Glenn St Hilaire, Weijie Yao, Elena Stamenova, Marie Hoeger, Sarah K. Nyquist, Valeriya Korchina, Kelcie Pletch, Joseph P. Flanagan, Ania Tomaszewicz, Denise McAloose, Cynthia Pérez Estrada, Ben J. Novak, Arina D. Omer, Erez Lieberman Aiden<br>bioRxiv 254797; doi: https://doi.org/10.1101/254797 McKinney, G. J., Waples, R. K., Seeb, L. W., & Seeb, J. E. (2017). Paralogs are revealed by proportion of heterozygotes and deviations in read ratios in genotyping-by-sequencing data from natural populations. <em>Molecular Ecology Resources</em>, <em>17</em>(4), 656-669. https://doi.org/10.1111/1755-0998.12613
description: # Data from: Rapid colonisation of synanthropic stone martens in a highly
 urbanised region: Insights from temporal and spatial analysis
 [https://doi.org/10.5061/dryad.3xsj3txrn](https://doi.org/10.5061/dryad.3xsj3txrn) ## Description of the data and file structure See the Methods section for information on the collection of samples, GBS analysis, SNP genotyping and filtering steps. The resulting filtered SNP data set holds genotypes of 376 individuals of *Martes foina* using 5536 SNPs. ### Files and variables #### File: metadata\_samples.csv **Description:** Sample information with sample ID, group ID, sex (empty cells represent gender not know), collection date (or year), and coordinates (Belgian Lambert 72 coordinate system). #### File: filtered\_snps.csv **Description:** Filtered SNP data file with 376 samples of stone marten and 5536 SNPs, with samples in rows and SNPs in columns. In the first two columns the sample ID and the group ID is provided. Missing genotypes are indicated with “NA”.