Population genetic studies in non-model systems increasingly use next-generation sequencing to obtain more loci, but such methods also generate more missing data that may affect downstream analyses. Here we focus on the Principal Component Analysis (PCA) which has been widely used to explore and visualize population structure with mean-imputed missing data. We simulated data of different population models with various total missingness (1%, 10%, 20%) introduced either randomly or biased among individuals or populations. We found that individuals biased with missing data would be dragged away from their real population clusters to the origin of PCA plots, making them indistinguishable from true admixed individuals and potentially leading to misinterpreted population structure. We also generated empirical data of the big brown bat (Eptesicus fuscus) using restriction site-associated DNA sequencing (RADseq). We filtered three data sets with 19.12%, 9.87%, and 1.35% total missingness, all showing nonrandom missing data with biased individuals dragged towards the PCA origin, consistent with results from simulations. We highlight the importance of considering missing data effects on PCA in non-model systems where nonrandom missing data are common due to varying sample quality. To help detect missing data effects, we suggest to 1) plot PCA with a color gradient showing per sample missingness, 2) interpret samples close to the PCA origin with extra caution, 3) explore filtering parameters with and without the missingness-biased samples, and 4) use complementary analyses (e.g., model-based methods) to cross-validate PCA results and help interpret population structure.

These are the filtered vcf data sets (bat1, bat10, bat20) used for empirical analyses in this study. The corresponding sample information (including museum catalogues) are provided in Table S1 of the journal's online supplementary material.