Molecular Ecology Resources

Molecular data have revolutionised taxonomic and ecological research on the hyperdiverse communities of aquatic benthic microinvertebrates known as meiofauna. However, reference sequence databases remain highly incomplete, with variable barcode genes or fragments studied from taxon to taxon. Furthermore, there is a typical tradeoff between universality of primers and phylogenetic resolution, with rRNA markers being robustly recoverable but failing to resolve species-level divergences, and mitochondrial markers showing the reverse trend. Here, we introduce Oxford Nanopore rRNA and COI amplicon sequencing (OrCa-seq), a rapid, low-cost protocol for parallel long-range PCR amplification and multiplexed sequencing of four amplicons, spanning the nearly-complete rRNA cistron ([~]7-8 kb) and the widely studied Folmer region of COI (represented as overlapping 313 and 658 bp amplicons). This protocol, with its associated bioinformatic workflow, was designed for conducting biodiversity inventories of meiofauna and can be easily carried out in field research and educational contexts, with data available from 96-well plates of specimens within a day of lysis. To validate the method, we processed six plates of student-isolated freshwater and limno-terrestrial meiofauna, characterising the recovery of target genes and taxa with both automated and human-curated BLAST database comparisons. These data demonstrate the universal applicability of OrCa-seq across effectively all meiofauna, including the very smallest species. Nonetheless, recovery efficiency for each amplicon shows variation by taxon, with the full-length Folmer COI amplicon standing out as the most challenging. We present exemplar phylogenetic trees integrating reference sequences, demonstrating the utility of these data in confirming morphological determinations and in identifying anonymous specimens in a reverse taxonomy context. While developed in a specific educational context for use on meiofauna, the OrCa-seq approach should be readily scalable to larger research datasets, adaptable to many specimen types, and to any combination of taxon-or target-specific primers. As such, it represents a compelling multi-locus extension to the ever-growing repertoire of nanopore DNA barcoding protocols.

O_LIEnvironmental DNA (eDNA) analyses have become a powerful tool for non-invasive biodiversity monitoring, yet the applicability of population genetic approaches to environmental samples remains largely unexplored. Even when genetic traces originate from a single individual, low target DNA concentrations and amplification or sequencing artefacts can compromise downstream genetic inferences. Here, we present a novel approach for obtaining demographic insights and lineage-level mitogenomic information from aquatic eDNA samples collected near vertebrate individuals. C_LIO_LIPaired eDNA and tissue samples were collected during sperm whale (Physeter macrocephalus) encounters in the Azores. Samples were screened for the presence of vertebrate eDNA and analyzed with a novel molecular sex identification assay. Additionally, long-range PCR was used to amplify up to five mitochondrial DNA fragments ([~]3-4k bp) before subsequent sequencing on an Oxford Nanopore Technologies platform. A stringent three-tier filtering framework capable of identifying true mitogenomic variation across eDNA samples was developed for maximum recovery of genetic diversity at the haplogroup level. By benchmarking eDNA samples via their paired tissues, parameter values were optimized to maximize concordance and minimize spurious variant calls. C_LIO_LISexing was successful for 50% of eDNA samples, with 96% concordance to paired tissues, and marine vertebrate DNA concentration significantly predicted sexing success. Further, Medaka polishing produced high identity mitochondrial consensus sequences (>16 kb) from eDNA samples. Across filtering regimes in the framework, curated SNP panels comprising up to 453 high-confidence mitochondrial SNPs resolved 19 haplogroups, with 93% concordance between eDNA and tissue samples. An intermediate bioinformatics filtering strategy maximized biologically accurate haplogroup recovery while minimizing sequencing artefacts, providing the most reliable lineage-level inferences. C_LIO_LIThis integrative approach demonstrates that targeted nuclear assays combined with long-range mitochondrial sequencing can recover individual-level genetic information from aquatic eDNA. By defining analytical thresholds governing success, the framework advances non-invasive genetic monitoring of populations via eDNA and enables population-level monitoring and conservation of endangered and genetically-vulnerable species. C_LI

DNA metabarcoding is a central tool in biodiversity research and monitoring, producing detailed taxa lists with comparatively little time and effort. One of its limitations, however, is the lack of quantitative data on biomass or abundance. This limitation has two main reasons: 1) template copy number variation and 2) primer-induced amplification bias. Many metabarcoding markers are mitochondrial and mitochondrial copy numbers vary in animal tissues, potentially decoupling sequence counts from biomass. Additionally, primer mismatches can lead to taxon-specific amplification biases, for which PCR cycle calibration has been proposed as a solution. To mechanistically study both effects, we constructed mock communities of different arthropod species. We combined digital droplet PCR and COI metabarcoding to quantify relationships between biomass, mitochondrial copy number and metabarcoding reads. Mitochondrial DNA copy numbers per biomass varied strongly within and among the different taxa. Metabarcoding reads did not reflect input mitochondrial DNA copies without a correction. Attempts to correct for amplification bias via PCR cycle calibration failed as read proportions remained stable across cycles. We therefore mathematically derived an approach to estimate relative amplification bias and initial mitochondrial DNA copy numbers in a sample based on a non-exponential amplification bias model and demonstrate its applicability. Still, the detected high variation in mitochondrial copy numbers and derived prerequisites necessary to calculate amplification efficiencies and mitochondrial copy numbers limit the practical application. Our study highlights fundamental constraints of quantitative metabarcoding and underscores the need for additional methodological approaches for quantitative insights while delivering essential conceptual insights.

Target enrichment methods have provided unprecedented advances in phylogenomics. Targeting hundreds of conserved regions has proven to be a good tradeoff between cost and efficiency, while being useful for museomics and diversified non-model clades. Unfortunately, current methods used for identifying such regions involve high degrees of conservation within targeted elements, usually pushing researchers to rely on flanking data with little guarantee for homology. With a growing number of high quality genomes available throughout the Tree of Life emerges new opportunities to improve marker selection. In this study, we introduce GABBI, a new method for designing target capture probes by taking advantage of genome alignments, avoiding the selection of a single reference genome that can cause notable biases. We compare GABBI-derived markers to the most commonly used probe design method, PHYLUCE, at two taxonomic scales, the weevil superfamily Curculionoidea and the tribe Pachyrhynchini. At both taxonomic scales, results show that our new method allows identifying more variable loci that prove to be more phylogenetically resolutive than the PHYLUCE-derived ones. Doing so, we provide the first probe set specifically designed for weevils, targeting a wide set of 4,255 shared homologous regions, encouraging future research on systematics and macroevolution of one of the most diverse and economically important groups of insects. By providing GABBI as an automated and open-access pipeline, we hope to open new probe design opportunities to other taxonomic groups that face similar phylogenetic obstacles.

Understanding evolutionary processes is greatly facilitated by high-quality data on genetic variation. We report the development of a genomic toolkit for a recently bottlenecked, long-term studied species, the Seychelles warbler (Ptimerl dezil; Acrocephalus sechellensis). This toolkit comprises a reference genome assembled into 31 chromosomes, together with functional annotations and reference-panel-free imputation of whole-genome sequences from 1,935 individuals. The genomic data have been used to assign the sequenced individuals into a genetic pedigree. Individual genomic data are associated with a suite of phenotypic metadata, amassed from three decades of fieldwork in this closed, long-term monitored population. We compared sex and parentage assigned using the genomic data with the previously recorded sex and parentage metadata to identify and correct 41 sample DNA samples labelled with the wrong identity. This population resource enables a wide range of analyses, that include, but are not limited to phylogenetics, metabarcoding, recombination rates, linkage patterns, adaptation, heritability, demographic history, selection, and inbreeding estimates. We wish to encourage interest from researchers seeking to collaborate on future analyses and data collection. Overall, our methods demonstrate the potential of next generation sequencing and statistical tools to provide dense genomic datasets at large sample sizes for wild populations.

Hybridization between invasive and native species poses a hidden but critical threat to biodiversity. While environmental DNA (eDNA) has revolutionized species monitoring, it has lacked the resolution to detect hybrid individuals. Here, we present the first experimental demonstration of hybrid identification using eDNA. Our method isolates a single cell in the environment (hereafter, eCell) and enables cellular-level analysis using multiplex digital PCR targeting nuclear markers from both parental species. Validation with controlled tank experiments using Oncorhynchus masou masou x Salvelinus leucomaenis leucomaenis hybrid individuals confirmed the methods ability to separately detect hybrid individuals from co-habiting purebred parent individuals. This eCell analysis overcomes the limitations of traditional eDNA methods and offers a scalable, non-invasive tool for detecting cryptic hybridization. By enabling early and accurate detection of hybrid individuals, it supports timely conservation decisions, including management prioritization and the protection of purebred populations. This novel technique bridges a critical gap in conservation genetics and enhances eDNAs utility for biodiversity management in the face of global change.

Microsatellite markers remain essential for individual-level genetic work in taxa where genome-wide methods are not yet routinely feasible due to extremely low DNA yields per specimen. In Gyrodactylus, even the most recent reference genomes have required pooling thousands of individuals, leaving a practical gap between genome-scale resources and individual-level analyses. Here we present a genome-informed microsatellite panel, developed by selecting single-copy loci with non-repetitive flanking regions and assembling all markers into a single multiplex PCR. Marker identity and performance were verified via amplification tests, Sanger sequencing, and cross-laboratory genotyping, confirming that the same samples generated identical fragment-size profiles in both laboratories. Long tandem repeats occasionally prevented exact repeat-count determination, yet allele-size classes were discrete and reproducible across replicates. The panel enables rapid individual identification and reliable strain and lineage assignment. It also offers a practical starting point for population-genetic and evolutionary studies that require individual-level data.

O_LIEffective conservation of highly migratory species requires understanding genetic structure across breeding populations and access high{square}resolution markers capable of assigning individuals from mixed aggregates (e.g. bycatch or new nesting sites) to their natal origins. Genomic approaches provide unprecedented resolution but add methodological challenges; thus, it is essential to first build a genomic baseline from known breeding areas and then evaluate strategies for assigning unknown individuals. C_LIO_LITo address this, we used 2b-RAD sequencing, a genomic reduction technique useful for degraded DNA, and loggerhead turtles as a case study. This species shows philopatric breeding, while juveniles and adults form mixed aggregations in foraging grounds. C_LIO_LIOur results highlight the importance of building baselines that include all potential source populations contributing to mixed aggregations. We detected hierarchical genetic differentiation and high resolution and successfully assigned the natal origin of 124 unknown individuals from four Mediterranean foraging grounds. These grounds showed distinct source contributions, and comparisons with previous studies suggest possible temporal shifts in stock composition. C_LIO_LIWe provide a comprehensive genomic baseline for individual assignment of Altanto-Mediterranean loggerhead turtles of unknown natal origin and a general framework for identifying population-specific threats in highly migratory species. C_LI

The coleoid cephalopods (octopus, cuttlefish, and squid) are emerging model organisms for neuroscience, development, and evolutionary biology. Determining their sex early in life is critical for population management and controlled experiments. Here, we present a protocol to non-invasively determine the sex of multiple cephalopod species as young as 3 hours post-hatching using a skin swab and quantitative PCR (qPCR). We describe steps for designing qPCR primers, swabbing live animals, extracting DNA, running the qPCR, and analyzing the results. For complete details on the use and execution of this protocol, please refer to Rubino et al.1 HighlightsO_LISwab live cephalopods as early as 3 hours post-hatching C_LIO_LIExtract DNA from cephalopod skin swabs C_LIO_LIPerform qPCR-based sex determination C_LIO_LIDesign and validate qPCR primers for new species C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/715692v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@3aa68dorg.highwire.dtl.DTLVardef@8c7e61org.highwire.dtl.DTLVardef@1bd45d9org.highwire.dtl.DTLVardef@134cc4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

The sunflower sea star (Pycnopodia helianthoides) suffered a catastrophic population decline across its range from 2013 to 2017 due to the devastating Vibrio pectenicida FHCF-3 driven sea star wasting disease (SSWD) pandemic with minimal signs of population recovery. The functional extinction of this apex predator across substantial parts of its range has created a need to identify and track the remaining intact populations. Environmental DNA (eDNA) approaches provide a simple, cost-effective, and non-destructive method for monitoring occurrences, and in some cases abundances, of marine species, consistently outperforming visual occurrence monitoring efforts in sensitivity, speed, and cost. Here, we designed, developed, and validated a P. helianthoides-specific eDNA assay to identify refugia, using both quantitative and digital droplet PCR approaches. We first generated the most comprehensive sea star mitochondrial genome reference database to date (n=93 taxa, n= 15 novel). We then used unikseq and Geneious bioinformatics software to identify the unique nad5 gene region and design a highly specific hydrolysis probe-based PCR assay. We validated the performance of this assay through laboratory, mesocosm, and field testing, demonstrating a highly specific and sensitive assay. In a field application of the new assay across regions in British Columbia, Canada, we found a positive correlation between P. helianthoides eDNA concentrations and biomass density, especially when appropriately accounting for spatiotemporal integration scales (R2=0.67). The eDNA assay provides a rapid and scalable tool for monitoring the sunflower sea star which has been proposed for listing as threatened under the U.S. Endangered Species Act of 1973. Molecular tools like the one presented here enhance management and recovery efforts not only by identification and monitoring of remnant wild populations, but also by helping to assess population level response and recovery following reintroduction efforts.

Obtaining large and well-resolved phylogenetic trees for neotropical clades is challenging, as many species inhabit remote regions, and sampling often relies on herbarium specimens with highly degraded DNA. Target capture provides an effective solution for retrieving molecular data from fragmentary material. However, data processing using tools generally designed for diploid organisms and single-copy loci is also challenging, particularly when events such as genome duplication and hybridisation have shaped the lineage evolution. We used dual-hybridisation to integrate Ochnaceae-specific and universal probes to reconstruct the phylogenetic relationships of Sauvagesieae, a pantropical clade with ca. 90 species mainly distributed in the northern Andes, the Brazilian Espinhaco Range, and the Amazon-Guyana region. We tested different filtering strategies involving missing data and paralogs to assess probable sources of tree discordance and topological uncertainty. We found no significant benefit in reducing tree discordance after removing entire genes due to the presence of paralogs or a high amount of missing data. Removing fragmentary sequences instead improved alignments and increased branch support of gene trees. By quantifying the proportion of SNPs, analysing the distribution of the allele frequencies, and gene-tree quartet frequencies, we found evidence of polyploidisation and hybridisation, which could reduce resolution at internal nodes, particularly in mountain clades. Our results underscore the importance of exploring the complexities of target-capture data, not only to improve phylogenetic resolution but also to understand the sources of phylogenetic conflict and the underlying molecular evolutionary processes.

Environmental DNA metabarcoding is a powerful monitoring tool for assessing aquatic biodiversity, as well as the sustainability and impacts of fisheries and aquaculture. However, conventional laboratory workflows remain time-consuming and dependent on dedicated infrastructures. Here, we present a field trial of a fully portable, off-grid eDNA metabarcoding pipeline that enables end-to-end analysis within a few days using compact equipment, including a BentoLab workstation and an Oxford Nanopore Technologies (ONT) MinION sequencer. The workflow was implemented during two international training courses in Norway and Brazil, where students and early career researchers collected environmental samples, extracted and amplified DNA, prepared DNA libraries, and sequenced on-site before performing bioinformatics and statistical analyses. In the case study detailed here, seven eDNA samples collected and analysed on-site in the Oslofjord allowed detection of 16 fish and elasmobranch species. Although overall diversity was lower than in earlier studies using Illumina-based sequencing, our protocol reliably detected key species and demonstrates that portable eDNA metabarcoding is feasible for rapid ecological assessment, surveillance of high-risk regions and/or deployment in remote or resourcelZllimited settings.

Pollen is a robust and widespread substance that captures a historical snapshot of a specific time and place, and it can be used to track movements through space by examining the pollen deposited on various objects. Palynology, the study of pollen, is used across fields such as conservation, natural history, and forensics, where it is particularly useful for tracing the origin and movement of objects. However, pollen has remained underutilized due to the difficulty of distinguishing many pollen taxa beyond the family level and limited pollen reference material to support location predictions. With recent developments in pollen DNA metabarcoding these issues have been rectified, but much of the available pollen data are primarily from wind-pollinated species, which are widespread and less informative of specific sample locations. Bee-collected pollen presents an untapped resource in training predictive models to geolocate sample origin. Here we compiled bee-collected pollen DNA sequence relative abundance data from three projects in the western U.S. and assessed the accuracy of supervised machine learning models to predict the location of sample origin based solely on pollen assemblage, without the need of incorporating additional data. Random Forest and k-Nearest Neighbors models yielded high accuracy across all projects. We also found that models trained on taxonomically clustered pollen assigned sequence variants (ASVs) performed slightly better than those trained on raw sequence data, but the difference was minor, indicating that models trained on raw sequence data can reliably predict location and avoid the time-consuming taxonomic assignment process. Our results demonstrate the utility of repurposing bee-collected pollen for geolocation and provide a framework for employing supervised machine learning in future geolocation efforts. HighlightsO_LIBee-collected pollen metabarcoding data was used to accurately predict sample origin C_LIO_LIRandom Forest and k-Nearest Neighbors algorithms were most accurate with lowest error C_LIO_LITaxonomically-classified and raw DNA sequence data training sets performed comparably C_LI

Airborne environmental DNA (eDNA) is a promising tool for detecting a wide range of taxa including threatened and invasive species, yet its application in management is constrained by a limited understanding of its temporal persistence, particularly in nature. We investigated the temporal persistence of airborne eDNA in a natural outdoor setting, using Bennetts wallaby Notamacropus rufogriseus as a case study. We captured airborne eDNA from a single Bennetts wallaby carcass, deployed in an area where wallabies are otherwise not present. A total of 180 samples were collected, spanning the period before deploying the carcass, the 11 days it was on site, and for 32 days after its removal, at distances of 1, 10, and 100 metres using both active (fan-assisted) and passive (no fan) collection methods. Although overall detection rates were low, wallaby DNA was detectable up to 100 metres shortly after the wallaby was introduced to the site and for up to three days after its removal. These findings indicate that airborne eDNA persists only briefly. Actively sampling air using battery-powered fans significantly improved detection rates relative to passive sampling. We demonstrate that airborne eDNA can detect individual organisms in outdoor environments, but reliable detection requires robust sampling and replication to capture rare, transient signals. By revealing how these signals persist over time, our findings provide a framework for optimizing field deployment and for distinguishing remnant DNA from new incursions.

AO_SCPLOWBSTRACTC_SCPLOWClose-Kin Mark-Recapture (CKMR) is a statistical framework for estimating demographic parameters of wild populations. Instead of recapturing individuals, it relies on the identification of closely-related pairs such as parents and offspring, or siblings. By measuring how often such close-kin are "recaptured" among sampled animals (whether alive or dead), scientists can estimate demographic parameters such as census size, mortality rates, and connectivity. CKMR is starting to change fisheries and wildlife management by giving more reliable demographic information, even for many species that resist conventional approaches. Here we introduce the kinference R package, which provides a set of tools for finding close-kin pairs among thousands of samples each genotyped at thousands of SNPs, and for associated quality control. The CKMR context implies different requirements and assumptions to many other kinship programs. In particular, kinference accounts empirically for linkage without requiring a genome assembly, is able to estimate and control false-negative and false-positive probabilities, and can cope with null alleles. The package has been developed and used in numerous CKMR projects since 2017. This paper documents the assumptions, statistical algorithms, and intended workflow for kinference.

The effective management of threatened shark populations relies on accurate demographic data, particularly operational sex ratios. While sex identification in intact shark bodies is straightforward through the presence of external male organs, namely claspers, it remains impossible for processed fins in the illegal wildlife trade, early-stage embryos in breeding programs, or archived tissue fragments and blood samples where morphological traits are lost. Here, we present a robust molecular sexing framework leveraging recently identified sequences from shark sex chromosomes, consistently organized in the XY system, to our current knowledge. Our approach consists of two distinct methodologies tailored to the the current identification status of sex chromosome sequences in the target species. For the whale shark Rhincodon typus and the brownbanded bamboo shark Chiloscyllium punctatum, we employed end-point PCR assays targeting male-specific Y-linked markers. For the cloudy catshark Scyliorhinus torazame, we developed a quantitative PCR (qPCR) assay targeting differential X chromosome dosage. In this dosage-based system, females (XX) are distinguished by an amplification profile approximately one cycle earlier than males (XY). By integrating X-linked dosage quantification, our framework provides a critical internal control that significantly enhances reliability, allowing researchers to distinguish true females from PCR failures. This toolkit offers a versatile solution for diverse applications, ranging from the study of sex determination mechanisms in pre-phenotypic embryos to the reconstruction of sex ratios from space-constrained tissue archives and global wildlife forensics, thereby contributing to the comprehensive conservation of shark biodiversity.

The marbled teal (Marmaronetta angustirostris) is a widely distributed but declining waterfowl species, classified as Near Threatened globally and Critically Endangered in Spain. Despite ongoing conservation actions, including ex situ management and population reinforcement programmes, the genomic consequences of long-term captivity, inbreeding, and patterns of functional genetic variation remain unknown due to the absence of a species-specific reference genome. Here, we present the first chromosome-level genome assembly for this species. The genome was generated using PacBio HiFi long reads and Omni-C data, yielding a 1.15Gb assembly with a scaffold N50 of 76.95Mb. A total of 97.16% of the assembly was anchored into 36 chromosome-scale scaffolds, including the Z and W sex chromosomes. BUSCO analysis recovered 99.2% of conserved avian genes. Gene prediction was performed using both ab initio and homology-based strategies, resulting in 16,048 protein-coding genes. This resource provides a foundation for genomewide analyses of inbreeding, demographic history, and adaptive variation, and will support evidencebased in situ and ex situ conservation strategies for this threatened species.

O_LIEnvironmental DNA (eDNA) metabarcoding is an emerging tool for biodiversity assessment in freshwater systems, offering high-resolution insights into community composition. Here, we apply eDNA metabarcoding to evaluate the ecological impacts of Eurasian beaver (Castor fiber) activity within a seminatural enclosure in the Scottish Highlands. C_LIO_LIWe collected seasonal water samples from nine sites, six influenced by beaver dams and three control sites with no evidence of beaver engineering, across a 40-hectare enclosure. Samples were analysed for vertebrate and macroinvertebrate diversity using established 12S and COI markers. C_LIO_LIVertebrate alpha diversity did not differ significantly between beaver and control sites, likely reflecting the small spatial scale and low species richness of upland Scottish streams. However, community composition differed significantly between treatments, especially for fish (PERMANOVA, R2 = 0.55, P < 0.001), with beaver-influenced sites dominated by three-spined stickleback and control sites by brown trout. Macroinvertebrate communities showed a 78% increase in gamma diversity in beaver-modified habitats relative to controls. Species composition varied strongly with beaver presence (PERMANOVA, R2 = 0.29, P < 0.001), likely due to the creation of lentic-lotic mosaics and associated microhabitat diversity. Seasonal variation was significant in both taxonomic groups, with the lowest species richness and highest community dispersion observed in summer, probably reflecting hydrological and temperature-driven dynamics in eDNA production and transport. C_LIO_LIOur findings reinforce previous evidence that beaver dam-building activity enhances beta diversity in headwater systems. Additionally, we demonstrate that eDNA metabarcoding is a sensitive method for detecting spatial patterns in freshwater biodiversity associated with these activities at scales ranging from tens to hundreds of meters. These approaches could inform future monitoring strategies aligned with landscape-scale beaver management and reintroductions. C_LI

The Japanese quail (Coturnix japonica) is a widely used model organism in developmental biology, genetics, and agriculture. Here, we present new, haplotyped, high-quality genome assemblies of the Japanese quail, generated using a combination of state-of-the-art sequencing technologies, including PacBio HiFi long reads, Oxford Nanopore sequencing, and Hi-C scaffolding. This assembly has a total length of 1.19 Gb, 80% of which is included in chromosomes, and is highly complete (BUSCO score aves_odb10: 97.3). Assembly metrics show a marked improvement in contiguity, with a significantly higher scaffold N50 and a lower number of contigs compared to the reference genome assembly. Remarkably, the assembly extends previously truncated chromosome ends, with 31 telomeres detected. In addition, we merged the existing Ensembl and Refseq annotations and obtained a combined set of 26,102 genes, of which 25,038 genes were successfully mapped on the improved assembly haplotype 1 (Cjap1.hap1). Together, these new genome assemblies and their enriched annotation provide a robust genomic framework for future research. They enhance our ability to investigate developmental processes, genetic and epigenetic inheritance, and host-pathogen interactions. Furthermore, they offer valuable insights for conservation genetics and sustainable breeding programs. This resource represents a critical step forward in leveraging the full potential of the Japanese quail as a model species in both basic and applied research.

Evidence supporting the use of airborne eDNA for biodiversity studies and ecosystem monitoring is growing. The promise of wide-area population dynamics data for downstream applications in targeted monitoring of pests and pathogens for agriculture and rare species for conservation is appealing; however, several technical challenges persist. Here, we focused on the development of a comprehensive dataset to facilitate assay development and accelerate the use of aerial sampling for species detection. Year-round metabarcoding data was generated using bacterial, fungal, plant, and arthropod primer sets and resulted in relative abundance estimates for 4,960 amplicon sequence variants (ASVs), 1,748 ASVs of which were assigned to a minimum taxonomic level of genus (bacteria, fungi, plants) or class (arthropods). Sequence diversity assessments and seasonal clustering based on presence/absence detection patterns were performed for individual ASVs, while discerning quantitative changes in seasonal abundance required grouping ASVs to at least the genus level. Examination of the technical aspects of metabarcoding suggested that the use of subsampling allows for consistent detection of genera with relative abundance values above 2 %, even when samples have varying sequencing depths. Sequencing depth was the primary determinate for detecting sporadic and/or rare ASVs. Sampler comparisons, common sources of variation, and the benefits of barcoding regional species to supplement the existing taxonomic databases were discussed. Insufficient knowledge of sampler coverage area for the different organism types was identified as a limitation to the deployment of aerial monitoring networks. Considerations for further aerial metabarcoding efforts are suggested based on our experimental findings. ImportanceOur study deals directly with the generation, analysis and limitations of airborne eDNA metabarcoding data for re-use by the broader environmental research community. This includes timing of seasonal detection for possible genera of interest across multiple kingdoms, including bacteria, fungi, plants and animals (specifically arthropods), and support for the generation of local databases to assess the current limitations of universal primers for species/genus taxonomic resolution. With regards to methodology, it continues to build upon established best practices for airborne eDNA collection in areas such as sub-sampling and sampling replicates, sampler type and sequencing depth. To accelerate possible uptake and application of the data, we provide the identified ASVs and their seasonal relative abundances as a resource.