DOI: 10.5061/dryad.g4f4qrg33
title: Genomic data reveals mixed support for the current subspecies designations in Eastern Screech-Owls (<em>Megascops asio</em>)

description: The Eastern Screech-Owl is a non-migratory resident forest owl that occurs
 across a broad distribution throughout much of eastern and central North
 America with five subspecies generally recognized across the range. Using
 reduced representation sequencing, we found that genetic differentiation
 among populations was generally low, with only two clear genetic clusters
 supporting that M. a. mccallii is distinct. Beyond these two main groups,
 we found subtle genetic clustering roughly corresponding to geographic
 region and the currently recognized subspecies, suggesting mixed support
 for current subspecies designations. To address these questions, we
 analyzed 228 individuals representing all five recognized subspecies at
 8,220 SNPs using standard population genetic approaches. In addition to
 the subtle populations structuring noted above, the genetically identified
 subpopulations also varied in estimated effective population sizes and
 metrics of genetic diversity. We also detected a weak, but significant,
 signal of Isolation by Distance across the range, suggesting clinal
 patterns of genetic variation across this broadly distributed species. The
 genetic population structure we uncovered broadens our understanding of
 subspecies-level taxonomic classification in this species and may be
 useful for effective conservation management if it becomes necessary.
description: # Genomic data reveals mixed support for the current subspecies
 designations in Eastern Screech-Owls (*Megascops asio*) Dataset DOI:
 [10.5061/dryad.g4f4qrg33](10.5061/dryad.g4f4qrg33) ## Description of the
 data and file structure We isolated genomic DNA using the DNeasy
 Extraction Kit (Qiagen, Valencia, California, USA) following
 manufacturer’s protocols, and quantified DNA concentrations using a Qubit
 2.0 fluorometer (Life Technologies, New York, USA). We generated
 double-digested restriction-site associated DNA markers (ddRADtags)
 following the protocol of Thrasher et al. (2018) with some modifications.
 For DNA digestion, we used the enzymes *Sbf*I and *Msp*I (New England
 Biolabs, Massachusetts, USA) and ligated ends of the digested genomic DNA
 using T4 DNA ligase (New England BioLabs) to P1 and P2 adapters (Peterson
 et al., 2012). Indexing groups were pooled in equimolar concentrations
 sequenced on a shared Illumina NovaSeqX lane (2 X 150bp paired end reads)
 by the Biotechnology Resource Center (BRC) Genomics Facility
 (RRID:SCR_021727) at the Cornell Institute of Biotechnology
 (http://www.biotech.cornell.edu/brc/genomics-facility). ***Variant Calling
 & Filtering*** We filtered raw sequences of 150 bp for quality using
 the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit). We removed
 sequences if a single base had a Phred quality score less than 10 and if
 more than 95% of bases had a Phred quality score less than 20. We used the
 process_radtags commands in STACKS 2.67 (Catchen et al. 2013) to
 demultiplex the remaining sequences, retaining reads only if they passed
 the Illumina chastity filter, contained an intact *Sbf*I RAD site, and did
 not contain Illumina sequencing adapters. ***Reference Genome Alignment***
 Using Bowtie2-build (Langmead & Salzberg 2012), we aligned each of the
 filtered and demultiplexed sequences to a scaffold-level *M. asio*
 reference genome (Robinson et al. 2025) which we had indexed for
 alignment. We used the ref_map.pl program from STACKS 2 version 2.67
 (http://catchenlab.life.illinois.edu/stacks/) which ran both the GSTACKS
 and POPULATIONS programs. We used the program POPULATIONS to filter
 genotypes and export the data. We required that a locus be present in a
 minimum of 80% of the individuals for it to be called and applied a
 minimum minor allele frequency filter of 0.05. Using the
 population.structure output from the POPULATIONS command, we analyzed the
 percentage of missing data for each sample and removed samples for which
 missing data was greater than 50%. We then filtered out samples where the
 adjusted mean GSTACKS coverage was less than 5x. These filtering steps
 followed standard STACKS 2 analysis protocol (Rivera-Colón & Catchen
 2022). ### Files and variables #### File: demult.sh
 **Description:** Script for demultiplexing pooled ddRAD libraries ####
 File: filter.sh **Description:** Script for filtering raw sequence data
 (trimming, QC) #### File: popmap_management **Description:** popmap file
 used in STACKS to generate the population-level VCF (individuals treated
 as populations based on Admixture data - see publication for details) ####
 File: popmap2 **Description:** popmap file used in STACKS to generate the
 range-wide VCF (all individuals treated as a single population - see
 publication for details) #### File: refmapSCRIPT **Description:** Script
 used in STACKS to map reads to an existing reference genome #### File:
 populations.snps.222align.vcf **Description:** Filtered VCF from stacks.
 Individuals were filtered and aligned to an existing screech-owl reference
 genome. This VCF can be used with either popmap file above to generate a
 range-wide or population-level data set ## Code/software For all of the
 data posted here, raw sequences were processed, filtered, and aligned in
 STACKS. Variants were also called using the STACKS pipeline. 
 
 Using the provided VCF, traditional downstream population genetic
 analyses can be conducted. ## Access information Other publicly accessible
 locations of the data: * 
 
 Data was derived from the following sources: *