{"title":"Test Species Discrimination in Codonopsis (Campanulaceae) Using Genome Skimming Data.","authors":"Chun-Jiao Li, Yin-Zi Jiang, De-Zhu Li, Qing-Zhe Wu","doi":"10.1111/1755-0998.70025","DOIUrl":"https://doi.org/10.1111/1755-0998.70025","url":null,"abstract":"<p><p>To overcome the limitations of conventional barcoding loci, plastid genome (plastome) and nuclear ribosomal DNA (nrDNA) sequences recovered from genome skimming, proposed as 'super-barcodes' have been suggested as candidates for delimitating recently diverged species or complex plant groups. DNA super-barcodes must be further assessed for their effectiveness in other diverse plant groups. This research focused on the genus Codonopsis, a medicinally significant yet taxonomically complex group characterised by morphological similarity and high phenotypic plasticity in response to environmental conditions. We analysed standard DNA barcodes and super-barcodes across 81 individuals from 36 of the 42 species of Codonopsis from Asia. Our work provides a comprehensive DNA barcode library for Codonopsis species identification. Our findings demonstrated that super-barcodes significantly improved the phylogenetic resolution and the discriminatory power compared to standard DNA barcodes. Since mitochondrial sequence variation is generally low in plant species, few studies have assessed its effectiveness as super-barcodes. We screened the mitochondrial protein-coding sequences (CDS) using genome skimming and evaluated the identification capacity of their combination. Unexpectedly, the discriminatory power of mitochondrial DNA with high nucleotide variation was comparable to that of the concatenated plastid CDS. However, the organelle genome cannot wholly determine the species boundaries of Codonopsis, which might be related to their rapid evolutionary radiation, ILS, hybridisation and strong natural selection. Future multi-locus nuclear markers will likely be developed in plants for additional discriminatory power. Our study provides new knowledge and insights into species discrimination of recently evolved Codonopsis taxa in a biodiversity hotspot.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":" ","pages":"e70025"},"PeriodicalIF":5.5,"publicationDate":"2025-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144803029","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sushma Reddy, Kristen Wacker, Mai Fahmy, Evon Hekkala, John M Bates, Steven M Goodman, Shannon J Hackett, Marie J Raherilalao, J Dylan Maddox
{"title":"VoronaGasyCodes: A Public Database of Mitochondrial Barcodes for Malagasy Birds.","authors":"Sushma Reddy, Kristen Wacker, Mai Fahmy, Evon Hekkala, John M Bates, Steven M Goodman, Shannon J Hackett, Marie J Raherilalao, J Dylan Maddox","doi":"10.1111/1755-0998.70027","DOIUrl":"https://doi.org/10.1111/1755-0998.70027","url":null,"abstract":"<p><p>Molecular tools are increasingly being used to survey the presence of biodiversity and their interactions within ecosystems. Indirect methods, like environmental DNA (eDNA) and invertebrate-derived DNA (iDNA), are dependent on sequence databases with accurate and sufficient taxonomic representation. These methods are increasingly being used in regions and habitats where direct detection or observations can be difficult for a variety of reasons. Madagascar is a biodiversity hotspot with a high proportion of endemic species, many of which are threatened or endangered. Here we describe a new resource, VoronaGasyCodes, a curated database of newly published genetic sequences from Malagasy birds. Our database is currently populated with six mitochondrial genes or DNA barcodes for 142 species including 70% of the birds endemic to the island and will be periodically updated as new data become available. We demonstrate the utility of our database with an iDNA study of leech blood meals where we successfully identified 77% of the hosts to species. These types of resources for characterising biodiversity are critical for insights into species distribution, discovery of new taxa, novel ecological connections and advancing conservation and restoration measures.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":" ","pages":"e70027"},"PeriodicalIF":5.5,"publicationDate":"2025-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144793103","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Brian J Johnson, Melissa C Graham, Elina Panahi, Carla Julia S P Vieira, Nisa Suraj Nath, Paul Mason, Jamie Gleadhill, Darran Thomas, Michael B Onn, Martin A Shivas, Damien Shearman, Jonathan M Darbro, Gregor J Devine
{"title":"An All-in-One Metabarcoding Approach to Mosquito and Arbovirus Xenosurveillance.","authors":"Brian J Johnson, Melissa C Graham, Elina Panahi, Carla Julia S P Vieira, Nisa Suraj Nath, Paul Mason, Jamie Gleadhill, Darran Thomas, Michael B Onn, Martin A Shivas, Damien Shearman, Jonathan M Darbro, Gregor J Devine","doi":"10.1111/1755-0998.70022","DOIUrl":"https://doi.org/10.1111/1755-0998.70022","url":null,"abstract":"<p><p>Next-generation sequencing (NGS) has the potential to transform mosquito-borne disease surveillance but remains under-utilised. This study introduces a comprehensive multi-loci metabarcoding-based MX (molecular xenomonitoring) approach to mosquito and arbovirus surveillance, enabling parallel identification of mosquito vectors, circulating arboviruses, and vertebrate hosts from bulk mosquito collections. The feasibility of this approach was demonstrated through its application to a large set (n = 110) of bulk field collections. This set was complemented by a number (n = 28) of single-species mosquito pools that had previously been screened for viruses using quantitative reverse transcription PCR (RT-qPCR) and metatranscriptomics. Universal alphavirus and flavivirus primer sets were used to screen for arboviruses in the resulting metabarcoding library. Viral amplicons were then indexed and combined with mosquito-specific (ITS2), universal invertebrate (COI), and vertebrate (Cyt b) barcode amplicons prior to sequencing. This approach confirmed the presence of all previously identified mosquito species, as well as those commonly misidentified morphologically, and enabled a degree of quantification regarding their relative physical abundance in each collection. Additionally, the developed approach identified a diverse vertebrate host community (18 species), demonstrating its potential for defining host preferences and, in tandem with the viral screens and associated vector data, understanding disease transmission pathways. Importantly, metabarcoding detected a diversity of regionally prevalent arboviruses and insect-specific viruses, with all three viral diagnostics demonstrating a similar sensitivity and specificity in detecting Ross River virus and Barmah Forest virus, Australia's most common arboviruses. In summary, multi-loci metabarcoding is an affordable and efficient MX tool that enables complete mosquito-borne disease surveillance.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":" ","pages":"e70022"},"PeriodicalIF":5.5,"publicationDate":"2025-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144774395","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
L. D. Parker, K. R. Todd, D. L. Nelson, N. Songsasen, M. G. Campana, W. J. McShea, M. Songer, T. Messerly, H. Shamon, J. E. Maldonado
{"title":"An Optimised Method to Identify Reintroduced Swift Foxes (Vulpes velox) Through SNP Genotyping of Non-Invasively Collected Scat Samples Using In-Solution Hybridisation Capture","authors":"L. D. Parker, K. R. Todd, D. L. Nelson, N. Songsasen, M. G. Campana, W. J. McShea, M. Songer, T. Messerly, H. Shamon, J. E. Maldonado","doi":"10.1111/1755-0998.70017","DOIUrl":"10.1111/1755-0998.70017","url":null,"abstract":"<div>\u0000 \u0000 <p>Genomic methods have become increasingly common in wildlife population studies over the past two decades. While noninvasive genetic sampling has been prevalent since the 1990s, the field has lagged in the adoption of high-throughput sequencing methods. In-solution hybridisation capture offers an efficient way to enrich and sequence degraded, low-quantity DNA with a large percentage of exogenous content (e.g., scat samples) for specific targets of interest. Despite their frequent use in ancient and historical DNA applications, hybridisation capture techniques have not been widely adopted for noninvasive genetic samples. Previous studies demonstrated that capture enrichment of single-nucleotide polymorphism (SNP) loci enables genotyping of field-collected kit fox and coyote scats to effectively identify species, individuals and sex. Here, we expanded on this work by (1) investigating whether probes designed for kit foxes can generate multi-locus SNP genotypes and identify sex in closely related swift foxes (<i>Vulpes velox</i>), (2) assessing the capability of the resulting genotypes to differentiate among swift foxes by calculating identity-by-state values between samples from the same individual, (3) exploring the impact of replicate index polymerase chain reactions (PCRs) on genotyping success, and (4) exploring the performance of the marker set for inference of population genetic structure. We applied these methods to samples from swift foxes reintroduced to the Fort Belknap Indian Reservation, Montana, and showed a success rate of 85%. Our developed methodology can be applied to monitor the success of swift fox reintroduction efforts by estimating dispersal, survival, and reproduction. We also showed that probes designed and optimised for one species can produce informative genotypes from closely related species, highlighting their versatility for broader applications in wildlife population studies.</p>\u0000 </div>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":"25 7","pages":""},"PeriodicalIF":5.5,"publicationDate":"2025-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144740759","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Effects of Vacuum-Heat-Assisted Sample Desiccation on Microbiome Surveys","authors":"Stilianos Louca, Claire E. Mullin","doi":"10.1111/1755-0998.70020","DOIUrl":"10.1111/1755-0998.70020","url":null,"abstract":"<p>Sample preservation in the field and during transport can be a logistical challenge for microbiome surveys, particularly in remote areas. Sample desiccation eliminates the need for complicated cold chains and dangerous preservatives. However, the effects of desiccation on modern microbiome workflows such as gene-centric metagenomic profiling and metagenome-assembled genome (MAG) binning, remain poorly understood. In addition, most common desiccation tools such as lyophilisation cannot easily be deployed in the field. Here, we describe a proof-of-principle sample desiccator using vacuum and heat, specifically built for deployment in the field and exhibiting low power consumption and cost. We then test the effects of vacuum-heat-assisted sample desiccation followed by storage at room temperature, in comparison to conventional freezing, on multiple soil and animal faecal samples, via metagenomic and 16S rRNA amplicon sequencing. We consider multiple metrics related to the success of DNA extraction, sequencing, contig assembly, OTU clustering, gene annotation and MAG construction, as well as effects on inferred microbial community composition. We find that the impact of drying on considered success metrics was almost always either minor, non-significant or positive. For a subset of source materials we observed moderate but statistically significant differences in terms of inferred microbial taxonomic and genetic composition. We conclude that vacuum- and heat-assisted desiccation can be a useful, practical and cost-effective tool for microbiome field surveys, when a high consistency with frozen samples is not required.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":"25 7","pages":""},"PeriodicalIF":5.5,"publicationDate":"2025-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1755-0998.70020","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144726278","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
John T Lovell, Rachel Walstead, Amelia Lawrence, Evan Stark-Dykema, Matthew C Farnitano, Avril Harder, Tomáš Brůna, Kerrie Barry, David Goodstein, Jerry Jenkins, Anna Lipzen, LoriBeth Boston, Jenell Webber, Mansi Chovatia, Joanne Eichenberger, Jayson Talag, Jane Grimwood, Jeremy Schmutz, John K Kelly, Andrea L Sweigart, Lila Fishman, John H Willis
{"title":"Comparative Analyses of Four Reference Genomes Reveal Exceptional Diversity and Weak Linked Selection in the Yellow Monkeyflower (Mimulus guttatus) Complex.","authors":"John T Lovell, Rachel Walstead, Amelia Lawrence, Evan Stark-Dykema, Matthew C Farnitano, Avril Harder, Tomáš Brůna, Kerrie Barry, David Goodstein, Jerry Jenkins, Anna Lipzen, LoriBeth Boston, Jenell Webber, Mansi Chovatia, Joanne Eichenberger, Jayson Talag, Jane Grimwood, Jeremy Schmutz, John K Kelly, Andrea L Sweigart, Lila Fishman, John H Willis","doi":"10.1111/1755-0998.70012","DOIUrl":"https://doi.org/10.1111/1755-0998.70012","url":null,"abstract":"<p><p>Yellow monkeyflowers (Mimulus guttatus complex, Phrymaceae) are a powerful system for studying ecological adaptation, reproductive variation, and genome evolution. To initiate pan-genomics in this group, we present four chromosome-scale assemblies and annotations of accessions spanning a broad evolutionary spectrum: two from a single M. guttatus population, one from the closely related selfing species M. nasutus, and one from a more divergent species M. tilingii. All assemblies are highly complete and resolve centromeric and repetitive regions. Comparative analyses reveal such extensive structural variation in repeat-rich, gene-poor regions that large portions of the genome are unalignable across accessions. As a result, this Mimulus pan-genome is primarily informative in genic regions, underscoring limitations of resequencing approaches in such polymorphic taxa. We document gene presence-absence, investigate the recombination landscape using high-resolution linkage data, and quantify nucleotide diversity. Surprisingly, pairwise differences at fourfold synonymous sites are exceptionally high-even in regions of very low recombination-reaching ~3.2% within a single M. guttatus population, ~7% within the interfertile M. guttatus species complex (approximately equal to SNP divergence between great apes and Old World monkeys), and ~7.4% between that complex and the reproductively isolated M. tilingii. Genome-wide patterns of nucleotide variation show little evidence of linked selection, and instead suggest that the concentration of genes (and likely selected sites) in high-recombination regions may buffer diversity loss. These assemblies, annotations, and comparative analyses provide a robust genomic foundation for Mimulus research and offer new insights into the interplay of recombination, structural variation, and molecular evolution in highly diverse plant genomes.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":" ","pages":"e70012"},"PeriodicalIF":5.5,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144688488","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Lucas Elliott, Frédéric Boyer, Teo Lemane, Inger Greve Alsos, Eric Coissac
{"title":"wholeskim: Utilising Genome Skims for Taxonomically Annotating Ancient DNA Metagenomes.","authors":"Lucas Elliott, Frédéric Boyer, Teo Lemane, Inger Greve Alsos, Eric Coissac","doi":"10.1111/1755-0998.70001","DOIUrl":"https://doi.org/10.1111/1755-0998.70001","url":null,"abstract":"<p><p>Inferring community composition from shotgun sequencing of environmental DNA is highly dependent on the completeness of reference databases used to assign taxonomic information as well as the pipeline used. While the number of complete, fully assembled reference genomes is increasing rapidly, their taxonomic coverage is generally too sparse to use them to build complete reference databases that span all or most of the target taxa. Low-coverage, whole genome sequencing, or skimming, provides a cost-effective and scalable alternative source of genome-wide information in the interim. Without enough coverage to assemble large contigs of nuclear DNA, much of the utility of a genome skim in the context of taxonomic annotation is found in its short read form. However, previous methods have not been able to fully leverage the data in this format. We demonstrate the utility of wholeskim, a pipeline for the indexing of k-mers present in genome skims and subsequent querying of these indices with short DNA reads. Wholeskim expands on the functionality of kmindex, a software which utilises Bloom filters to efficiently index and query billions of k-mers. Using a collection of thousands of plant genome skims, wholeskim is the only software that is able to index and query the skims in their unassembled form. It is able to correctly annotate 1.16× more simulated reads and 2.48× more true sedaDNA reads in 0.32× of the time required by Holi, another metagenomic pipeline that uses genome skims in their assembled form as its reference database input. We also explore the effects of taxonomic and genomic completeness of the reference database on the accuracy and sensitivity of read assignment. Increasing the genomic coverage of the genome skims used as reference increases the number of correctly annotated reads, but with diminishing returns after ~1× depth of coverage. Increasing taxonomic coverage clearly reduces the number of false negative taxa in the dataset, but we also demonstrate that it does not greatly impact false positive annotations.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":" ","pages":"e70001"},"PeriodicalIF":5.5,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144673544","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"RETRACTION: Age Prediction of Green Turtles With an Epigenetic Clock","authors":"","doi":"10.1111/1755-0998.70016","DOIUrl":"10.1111/1755-0998.70016","url":null,"abstract":"<p><b>RETRACTION</b>: B. Mayne, W. Mustin, V. Baboolal, F. Casella, K. Ballorain, M. Barret, M. A. Vanderklift, A. D. Tucker, D. Korbie, S. Jarman and O. Berry, “Age Prediction of Green Turtles With an Epigenetic Clock,” <i>Molecular Ecology Resources</i> 22, no. 6 (2022): 2275-2284, https://doi.org/10.1111/1755-0998.13621.</p><p>The above article, published online on 15 April 2022 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editors-in-Chief, Shawn Narum and Ben Sibbett; and John Wiley & Sons Ltd. The retraction has been agreed upon following an alert from one of the authors, S. Jarman, identifying that the results shown in Figure 2 are inaccurate and do not represent a real relationship between age and DNA methylation. Consequently, the editors consider the results and conclusions invalid. The authors, S. Jarman, B. Mayne, M. A. Vanderklift, O. Berry, K. Ballorain and M. Barret agree with the decision to retract this article.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":"25 7","pages":""},"PeriodicalIF":5.5,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1755-0998.70016","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144648082","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Lydia Hildebrand Furness, Richard Sabin, Marianne Strand Torvanger, Oliver Kersten, James H. Barrett, Bastiaan Star
{"title":"Historical Collections of Tropical Marine Mammals Are an Excellent Resource for Ancient DNA","authors":"Lydia Hildebrand Furness, Richard Sabin, Marianne Strand Torvanger, Oliver Kersten, James H. Barrett, Bastiaan Star","doi":"10.1111/1755-0998.70015","DOIUrl":"10.1111/1755-0998.70015","url":null,"abstract":"<div>\u0000 \u0000 <p>The ability to predict ancient DNA sequencing success in natural history collections is critical to reducing the amount of destructive sampling of a finite resource. So far, studies investigating such success have predominantly focused on taxa with ranges restricted to temperate or cold environments at northern latitudes, which likely aids DNA preservation. Here, we report remarkably high aDNA sequencing success in <i>Sirenia</i>, herbivorous marine mammals of which the distribution is currently constrained to the global tropics. We investigate 91 samples from 85 specimens comprising all four contemporary species and one extinct species, comparing different sample types (cranial/post-cranial bone, skin and cartilage), species, collections, and material age. We obtained remarkably high (e.g., > 20%) endogenous DNA preservation for the majority (e.g., ~57% percent) of samples. Sequencing success was linked to sample type, with cranial bones (including petrous and tympanic bones) yielding significantly higher endogenous DNA. Additionally, we obtained variable, but potentially superior DNA results for preserved cartilage and hide samples that can be associated with historical bone. Although such tissue is not always present, this type of material is easy to sample, with very limited destructive impacts on the associated bones, and we therefore highlight its untapped potential as a source of DNA. Overall, our results show the high success of ancient DNA retrieval from historical collections of species with a tropical distribution, expanding on the types of specimens that are available for temporal genomic analyses.</p>\u0000 </div>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":"25 7","pages":""},"PeriodicalIF":5.5,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144648080","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Andy Lee, William Hemstrom, Natalie Molea, Gordon Luikart, Mark R. Christie
{"title":"‘Highly-Informative’ Genetic Markers Can Bias Conclusions: Examples and General Solutions","authors":"Andy Lee, William Hemstrom, Natalie Molea, Gordon Luikart, Mark R. Christie","doi":"10.1111/1755-0998.70011","DOIUrl":"10.1111/1755-0998.70011","url":null,"abstract":"<p>High-grading bias is the overestimation power in a subset of loci caused by model overfitting. Using both empirical and simulated datasets, we show that high-grading bias can cause severe overestimation of population structure, and thus mislead investigators, whenever highly informative or high-<i>F</i><sub><i>ST</i></sub> markers are chosen (i.e., ascertained) and used for subsequent assessments, a common practice in population genetic studies. This problem can occur in panmictic populations with no local adaptation<i>.</i> Biased results from choosing high-<i>F</i><sub><i>ST</i></sub> markers may have severe downstream implications for management and conservation, such as erroneous conservation unit delineation, which could squander limited conservation resources to protect incorrectly defined ‘populations’. Furthermore, we caution that high-grading is not limited to <i>F</i><sub><i>ST</i></sub> approaches; high-grading bias is a concern whenever a small subset of markers are first chosen to explain differences among groups based on their degree of difference and are subsequently reused to estimate the degree of difference among those groups. For example, selecting high <i>F</i><sub><i>ST</i></sub> loci for use in a GT-seq panel or using differentially expressed genes to plot sample membership in multivariate space can both result in spurious structure when none exists. We illustrate that using statistically based outlier tests in place of arbitrary <i>F</i><sub><i>ST</i></sub> cut-offs can reduce bias. Alternatively, permutation tests or cross-evaluation can be used to detect high-grading bias. We provide an R package, PCAssess, to help researchers detect and prevent high-grading bias in genetic datasets by automating permutation tests and principal component analyses (https://github.com/hemstrow/PCAssess).</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":"25 7","pages":""},"PeriodicalIF":5.5,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1755-0998.70011","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144606958","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}