Dealing with assumptions and sampling bias in the estimation of effective population size: A case study in an amphibian population

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 geneti..., 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 (elutio..., , # 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

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