Cryptic hybridization between the ancient lineages of Natterer's bat (Myotis nattereri)

Studying hybrid zones that form between morphologically cryptic taxa offers valuable insights into the mechanisms of cryptic speciation and the evolution of reproductive barriers. Although hybrid zones have long been the focus of evolutionary studies, the awareness of cryptic hybrid zones increased recently due to rapidly growing evidence of biological diversity lacking obvious phenotypic differentiation. The characterization of cryptic hybrid zones with genome-wide analysis is in its early stages and offers new perspectives for studying population admixture and thus the impact of gene flow. In this study, we investigate the population genomics of the Myotis nattereri complex in one of its secondary contact zones, where a putative hybrid zone is formed between two of its cryptic lineages. By utilizing a whole genome shotgun sequencing approach, we aim to characterize this cryptic hybrid zone in detail. Demographic analysis suggests that the cryptic lineages diverged during the Pliocene,..., DNA extraction
DNA was extracted either by using QIAGEN DNeasy Blood and Tissue kit following the manufacturer’s recommendation or with a salt-chloroform extraction method  (Dietz et al., 2016; Müllenbach et al., 1989). Tissue samples were digested in a lysis buffer for one hour at 56°C followed by 37°C overnight. In the end, DNA was eluted in 90 µl of AE buffer (Qiagen). We checked the DNA concentration and purity by using a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc). The degree of DNA degradation was visualized by agarose gel (1%) electrophoresis.
For genomic library preparation 500 ng of DNA was mechanically sheared by a S220 Focused-ultrasonicator (Covaris Inc) to achieve a fragment length below 800 bp. The fragment size distribution of sheared DNA was checked with Bioanalyzer (High Sensitivity DNA Chip, Agilent Inc) and concentration was determined using Qubit 2.0 Fluorometer and dsDNA HS Assay Kit (Thermo Fisher Scientific Inc). 80 ng of fragmented DNA was used to p..., , # VCF file and BAM files

[https://doi.org/10.5061/dryad.msbcc2g50](https://doi.org/10.5061/dryad.msbcc2g50)

VCF file used for the analyses in the manuscript and 3 bam files that were used for the PSMC analysis.

## Description of the data and file structure

**all_samples_all_scaffolds_filtered.vcf.gz**

Variant calls file used for the population structure analysis. We used Freebayes v1.3.1 (Garrison & Marth, 2012). Only reads with a mapping quality score above 10 were used to generate a multi-sample VCF file. Subsequently, we filtered with VCFtools v0.1.17 (Danecek et al., 2011) using the following parameters: only biallelic sites, coverage per site between 2 and 40 for each individual, and mean coverage between 5 and 60 over all included individuals. Minimal mapping quality was set to 20, and we allowed for 10% missing data per site. Finally, indels were removed and the minor allele frequency threshold was set to 0.03.

**9992_psmc.bam**

BAM file from a southern Italian populat...