DOI: 10.5061/dryad.j0zpc86ps
title: Data from: Dealing with assumptions and sampling bias in the estimation of effective population size: A case study in an amphibian population

description: Accurately estimating effective population size (Ne) is essential for
 understanding evolutionary processes and guiding conservation efforts.
 This study investigates Ne estimation methods in spatially structured
 populations using a population of moor frog (Rana arvalis) as a case
 study. We assessed the behaviour of Ne estimates derived from the linkage
 disequilibrium (LD) method as we changed the spatial configuration of
 samples. Moor frog eggs were sampled from 25 breeding patches (i.e.,
 separate vernal ponds, ditches or parts of larger fens) within a single
 population, revealing an isolation-by-distance pattern and a local spatial
 genetic structure. Varying buffer sizes around each patch were used to
 examine the impact of sampling window size on the estimation of effective
 number of breeders (Nb). Our results indicate a downward bias in LD Nb
 estimates with increasing buffer size, suggesting an underestimation of
 Nb. The observed bias is attributed to LD resulting from including
 genetically divergent individuals (mixture-LD) confounding LD due to
 drift. This emphasises the significance of considering even subtle spatial
 genetic patterns. The implications of these findings are discussed,
 emphasising the need to account for spatial genetic structure to
 accurately assess population viability and inform conservation efforts.
 This study contributes to our understanding of the challenges associated
 with Ne estimation in spatially structured populations and underscores the
 importance of refining methodologies to address population-specific
 spatial dynamics for effective conservation planning and management.
description: The study site of c. 200 ha is part of the nature reserve and
 military domain ‘Klein Schietveld’ in Kapellen near Antwerp, Belgium
 (51.358 N, 4.495 E; Fig. S1). In March 2017, heathland pools, fens and
 temporary ponds were screened for the presence of egg clutches possibly
 belonging to moor frogs. In total, eggs were sampled in 26 locations where
 clusters of clutches were found. These locations consisted of separate
 vernal ponds, ditches or parts of larger fens; they are called ‘breeding
 patches’ from now on. In each breeding patch, up to 50 intact and
 distinguishable clutches were sampled and three eggs per clutch were
 taken. The eggs were stored in pond water in a refrigerator until
 DNA-extraction (maximally a few days after sampling). DNA-extraction was
 performed on two eggs per clutch. The jelly coats were first removed using
 a scalpel. DNA was extracted from the embryo’s using DNeasy Blood
 & Tissue Kit (Qiagen) with a lysis step of one hour and eluted in
 70 μl AE buffer (elution performed twice). The integrity of DNA of 10 % of
 the samples was assessed on 1 % agarose gels, while the DNA concentration
 of all tissue samples was measured with Quant-iT Picogreen dsDNA Assay Kit
 (Invitrogen, Thermo Fisher Scientific) using a Synergy HT plate reader
 (BioTek). In order to make the distinction between samples from two
 different species, <em>Rana arvalis</em> and <em>R.
 temporaria</em>, DNA from one egg per clutch and from all larvae was
 analysed with the RFLP method of Palo and Merilä (2003).
 The <em>Rana arvalis </em>eggs were<em>
 </em>genotyped at 19 microsatellite markers via multiplex PCR and
 genotyping analysis on an ABI 3500 Genetic Analyzer. Allele calls were
 scored using the GeneMapper v4.1 software with fragment sizes based on
 GeneScan 600 LIZ Size Standard. Negative controls were included in each
 96‐well PCR to allow for detection of reagent contamination.
 Reproducibility was evaluated using 3 % blindly replicated samples, two to
 five times within and across well plates. One reference sample was further
 added to each well plate. Samples with genotypes for less than 50 % of the
 loci were reanalysed or replaced with genotypes of eggs of the same clutch
 where possible. The average error rate per locus was
 1%. Three markers, Rtempμ4, Rtempμ5 and Rt2Ca2-22, showed no polymorphism
 and alleles of locus Rtempµ9 could not be identified unambiguously. Locus
 RECALQ showed deviations from Hardy-Weinberg equilibrium (HWE) in 5
 breeding patches and proportions of null alleles higher than 0.20 in at
 least 10 breeding patches. Also, RlatCa41 deviated from HWE in 7 breeding
 patches and null alleles in at least 6 patches. Both markers were excluded
 from further analysis.
description: # Data from: Dealing with assumptions and sampling bias in the estimation
 of effective population size: A case study in an amphibian population
 [https://doi.org/10.5061/dryad.j0zpc86ps](https://doi.org/10.5061/dryad.j0zpc86ps) ## Description of the data and file structure genotypes_eggs_moor_frog.csv: the microsatellite genotypes of *Rana arvalis* eggs collected in Klein Schietveld, Belgium in March 2017. The multilocus genotypes for 13 microsatellite markers of 729 *R. arvalis* eggs collected from 366 clutches in 25 breeding patches. In the first five columns the following information is provided: the sample ID, the ID of the breeding patch, the egg clutch ID, followed by the coordinates of the breeding patch (Belgian Lambert 72 coordinate system). The next 13 columns represent the different microsatellite markers with the loci names mentioned in the column headers. Alleles are separated by a forward slash. Missing genotypes are indicated with “NA/NA”. ### Files and variables #### File: genotypes\_eggs\_moor\_frog.csv **Description:**  ##### Variables * Sample: sample ID for each sampled egg * breeding patch: ID for every breeding patch (i.e., locations consisting of separate vernal ponds, ditches or parts of larger fens) * clutch ID: ID for every sampled egg clutch of Rana arvalis * X: X-coordinate in Lambert 72 coordinate system (Easting) * Y: Y-coordinate in Lambert 72 coordinate system (Northing) * RA14: microsatellite locus, alleles are separated with a forward slash * RC08604: microsatellite locus, alleles are separated with a forward slash * RCIDII: microsatellite locus, alleles are separated with a forward slash * RNTYR2: microsatellite locus, alleles are separated with a forward slash * RtsB14: microsatellite locus, alleles are separated with a forward slash * RA03: microsatellite locus, alleles are separated with a forward slash * RA04: microsatellite locus, alleles are separated with a forward slash * RA11: microsatellite locus, alleles are separated with a forward slash * RlatCa18: microsatellite locus, alleles are separated with a forward slash * RRD590: microsatellite locus, alleles are separated with a forward slash * RtP: microsatellite locus, alleles are separated with a forward slash * RA13: microsatellite locus, alleles are separated with a forward slash * RtU4: microsatellite locus, alleles are separated with a forward slash ## Code/software The file is a comma delimited csv, which can be opened in many spreadsheet programs and data visualisation tools.