Advancements in historical genomics increasingly leverage museum collections to study past ecosystems, species interactions and biodiversity. Formalin-fixed, ethanol-preserved specimens, once thought inaccessible to molecular analyses due to DNA degradation, are emerging as valuable genomic resources. If recoverable and reliably attributable, DNA within preservation media could provide a non-destructive alternative to conventional tissue sampling, with the potential to expand molecular access to valuable or irreplaceable specimens. We tested whether preservation media contains recoverable DNA suitable for taxonomic inference. We coupled passive adsorption and active filtration of specimen media with hot alkaline lysis DNA extraction followed by metabarcoding and shotgun metagenomics. DNA was recoverable across samples, including 41 of 61 (~67%) targets in a composite sample. However, detections were dominated by non-target taxa, indicating that preservation media retain a layered mixture of specimen-derived DNA and broader collection-level background. Detection success tracked with preservation chemistry (near-neutral pH and low residual formaldehyde) rather than specimen age. Method choice influenced detections: active filtration increased target detections but admitted more background; passive capture was sparser but more selective; shotgun sequencing retrieved broader vertebrate signals, including reptiles, but was heavily enriched for non-targets. Because both target and non-target taxa were often abundant, read-abundance cut-offs were unreliable for attribution. Spirit-media DNA is therefore best interpreted as a collection-level signal and a screening tool to identify jars with molecular potential (e.g., taxa of conservation or biosecurity interest), rather than as a definitive non-destructive proxy for specimen identity. Prioritising chemically favourable jars and implementing rigorous contamination controls should improve signal interpretability and help unlock the value of preservation media for historical genomics.

Genetic reference databases underpin a wide range of molecular approaches used to study cetacean biodiversity, including environmental DNA (eDNA), yet their reliability depends critically on data completeness, taxonomic accuracy, and metadata quality. Here, we present the first global assessment of mitochondrial sequence availability for cetaceans, evaluating taxonomic coverage, geographic representation, metadata completeness, and the distribution of five commonly targeted mitochondrial markers (12S rRNA, 16S rRNA, D-loop, cytochrome oxidase I, and cytochrome b). We retrieved 17,569 cetacean accessions from the NCBI Nucleotide database and an additional 259 COI-only records from BOLD Systems. Sequence availability was strongly biased toward Delphinidae and Balaenopteridae, whereas several families, notably Ziphiidae, were markedly underrepresented. We also identified discrepancies between database records and currently accepted cetacean taxonomy (e.g., outdated genera, non-accepted species, and collapsed higher-level taxa). Among markers, the D-loop dominated database representation, largely as standalone sequences (12,437 records), reflecting historical and current sequencing priorities and underscoring its continued relevance for population-level studies and eDNA marker development. Only 38% of accessions included geographic metadata, with georeferenced records concentrated primarily in the Americas and the Northwest Pacific, while large regions, including much of Africa, remained poorly represented. Although broad geographic patterns mirrored known family distributions, pronounced regional and taxonomic gaps persist. Our results highlight critical deficiencies in mitochondrial reference databases for cetaceans and emphasise the need for improved metadata standards, targeted sequencing of underrepresented taxa and regions, and open data sharing to enhance the effectiveness and global applicability of eDNA-based cetacean monitoring.

Studies elucidating the molecular basis and evolutionary consequences of karyotypic changes in mammals remain scarce. Here, we investigate chromosomal evolution by focusing on two contrasting lineages within the family Vespertilionidae (Chiroptera): the karyotypically variable tribe Pipistrellini and the highly conserved genus Myotis. Pipistrellini exhibits extensive karyotype diversity, with diploid numbers (2n) ranging from 26 to 44, whereas Myotis demonstrates remarkable stability, maintaining 2n = 44 across nearly all studied species. To uncover the mechanisms driving these divergent evolutionary trajectories, we generated a high-quality chromosome-level genome assembly for Pipistrellus abramus (2n = 26). By integrating multiple high-quality vespertilionid genomes, we reconstructed the family phylogeny and inferred an ancestral karyotype of 2n = 44, revealing fusions and fissions as the primary drivers of karyotypic diversification. We further identified an enrichment of rolling-circle (RC) and recent DNA transposons in genes involved in DNA metabolism, suggesting a mechanistic basis for transposable element (TE) tolerance in Vespertilionidae. In P. abramus, a derived chromosome originated from three ancestral chromosomes via Robertsonian and end-to-end fusions, with TEs significantly enriched at fusion sites. Genome stability-related genes and contracted gene families also appear to facilitate adaptive responses to structural changes. These findings provide novel insights into the molecular mechanisms underlying chromosome evolution and speciation in mammals.

Environmental DNA (eDNA) metabarcoding can rapidly characterise biodiversity, yet its accuracy and effectiveness are limited by incomplete DNA barcode reference databases. We evaluated how comprehensive reference databases that include sequence variation within genomes (intragenomic) and across individuals and species (intergenomic) improve eDNA-based biodiversity assessments. We collected coral tissue and water samples at deep sites offshore Puerto Rico for reference barcoding and eDNA metabarcoding. Genome skimming coral specimens yielded 28S barcodes for 314 of 346 samples (90.8%) and revealed divergent intragenomic 28S lineages in multiple octocoral families. Incorporating local reference barcodes substantially changed ASV taxonomic classifications: 22 ASVs (8.9%) gained genus-level resolution, 19 ASVs (7.7%) were reassigned to different genera, and 14 ASVs (5.7%) lost incorrect genus-level classifications. Thus, incomplete reference databases produce not only unclassified ASVs but also false positive detections and ecologically meaningful misclassifications. When intragenomic 28S lineages were excluded from the reference database, 18 ASVs (7.4%) could not be classified to family or genus, demonstrating that unrecognised intragenomic variation can be mistaken for unsampled taxa. Integrating reference genome skimming and eDNA metabarcoding expanded known coral family richness by 36% at depths shallower than 1000 m and by 181% at depths greater than 1000 m. eDNA also detected two coral families previously unknown off Puerto Rico and nearby islands, underscoring its potential for biodiversity discovery.

Faeces serve as a widely used non-invasive material in wildlife studies, from which host DNA can be enriched for high-throughput sequencing. However, the characteristics of faecal DNA sequencing, particularly the relationship between sequencing volume and genotyping accuracy or genomic coverage, have remained unclear. This knowledge gap makes it challenging to pre-estimate the required sequencing depth to ensure data quality while controlling costs. In this study, we used host-enriched faecal DNA obtained via peri-extraction enrichment by SDS (PEERS) to perform comparative analyses of whole-genome sequencing data from paired faecal and blood samples of 11 captive Amur tigers. We systematically evaluated how data volume affects genomic coverage and genotyping accuracy. Our results revealed that faecal sequencing reads cover the genome unevenly, which may compromise the reliability of underrepresented allele identification and subsequent genomic analyses. Specifically, genomic coverage and genotyping accuracy, including precision, error rate, recall and F1-score, improved with increasing sequencing depth in both blood and faecal DNA. For reliable genotyping (values > 0.95), a minimum average depth of 20× was required for blood-derived DNA, whereas faecal DNA needed up to 50× depth when the nuclear DNA enrichment efficiency (EEN, defined as the ratio of nuclear to bacterial DNA copy number) reached 5E-5. At this sequencing volume, population-level indices, such as nucleotide diversity (π), polymorphism information content (PIC), the distribution of π in 500-kb sliding windows and SNP density, as well as individual-level indices, including heterozygosity, kinship coefficients, inbreeding coefficient (FROH) and genetic load, were highly consistent between faecal and blood samples. Only observed heterozygosity (Ho) and kernel density distributions of minor allele frequency (MAF) showed slight discrepancies. These findings underscore that effective host DNA enrichment using PEERS combined with deep sequencing is essential to achieve adequate genomic coverage and accurate genotyping when utilising faecal samples. Monitoring enrichment efficiency can help determine the necessary sequencing depth, facilitating cost-effective experimental design. This study offers practical guidance for the application of faecal DNA in genomic research and supports the broader use of this non-invasive material in future wildlife studies.

ABSTRACTDNA extraction with an alkaline buffer system called ‘HotSHOT’ is widely used for barcoding because it is rapid, inexpensive, and voucher preserving, but it remains unclear whether sufficient genomic DNA (gDNA) remains in small vouchers for downstream use in genomics. We here evaluate gDNA quality and quantity before and after HotSHOT treatment of 11 insect families representing six orders. Some specimens were flash frozen immediately after collection, while others were kept for 1 week at tropical temperatures in ethanol to mimic Malaise trap conditions. Encouragingly, we show that gDNA of sufficiently high quality and quantity for genomic sequencing remained in specimens treated with HotSHOT. We also show that DNA integrity was strongly influenced by field storage, with specimens exposed to Malaise trap conditions showing such pronounced degradation that the standard HotSHOT treatment no longer significantly altered DNA quality. For control material, HotSHOT treatments involving longer exposure to high temperature led to smaller fragment lengths, with the effect apparently being influenced by the degree of specimen sclerotization. Our results thus suggest that optimised HotSHOT treatments, together with carefully controlled pre‐extraction storage, preserve voucher gDNA of sufficient quality for downstream genomic analyses with both short‐read and possibly even some long‐read sequencing technologies. We provide protocol selection guidelines that improve voucher gDNA preservation in HotSHOT‐treated samples. This is particularly important for many species which are only known from one or few specimens discovered during barcoding projects.

ABSTRACTAquatic ecosystems host diverse organisms across all six life kingdoms, yet their complex interactions remain poorly understood, primarily due to limitations in transkingdom species detection methods. To address this limitation, we developed HiMBar (https://github.com/Xchenkai2019/HIFI_barcoding), a high‐fidelity (HiFi) metagenomic barcoding approach that utilises long, highly accurate reads to extract multiple full‐length marker genes (such as rRNA genes, COI, rbcL) directly from environmental DNA sequencing reads. These genes are subsequently clustered into operational taxonomic units (OTUs) for species identification, eliminating the need for PCR amplification or sequence assembly. HiMBar outperforms existing DNA‐based methods in accuracy, recall and consistency. Applying HiMBar, we identified a stable interaction network among Cyanobacteria, Planctomycetota, Verrucomicrobiota and Fungi. Further analysis revealed that glucose metabolism plays a key role in maintaining these interactions. Our study offers a powerful tool for transkingdom species monitoring and provides a case study for exploring transkingdom interactions and their molecular mechanisms.

ABSTRACTDemographic information, such as sex ratios, is essential for understanding population dynamics and informing conservation strategies. Yet obtaining sex ratios in natural populations can be challenging due to logistical, ethical and legal constraints. Environmental DNA (eDNA) has revolutionised non‐invasive biodiversity monitoring, but its potential for assessing demographic parameters remains largely unexplored. Here we present an eDNA‐based method to monitor sex ratios of populations by quantifying sex‐specific SNP alleles. Using RADseq data from Balkan crested newts (Triturus ivanbureschi), we identified a male‐specific allele that was consistently present in all males and absent in females. We then developed a Droplet Digital PCR (ddPCR) assay to quantify allele ratios and validated it on mock (DNA extract mixtures) and eDNA samples with known sex ratios. Our sex‐specific SNP assay successfully distinguished male‐ and female‐biassed ratios in mock samples and showed a strong positive relationship between the proportion of males and the male‐specific allele. While resolution was lower in eDNA samples, sex ratio estimates reflected population composition, particularly when corrected for biomass. Performance was mainly influenced by inter‐individual variation in male allele copy numbers, but this effect diminished as the number of males increased, reflecting natural populations better. For effective field application, maximising nuclear eDNA recovery, validating marker specificity and accounting for species‐specific life history traits when sampling will be crucial. With further field validation, our eDNA‐based method could support large‐scale, non‐invasive sex ratio monitoring, offering valuable insights into species phenology and population dynamics to guide conservation efforts.

Establishing best practices for the overall workflow of environmental DNA (eDNA) sampling is necessary to increase reproducibility and precision in estimates of biodiversity across studies. Rigorous comparisons between eDNA sample preservation strategies for long-term storage durations are lacking, and previous studies have primarily evaluated DNA yield rather than detection success, despite detection being of critical importance when studying rare or elusive species. Here, we assessed the efficacy of common preservation media, storage temperatures and DNA extraction methods on eDNA yield and detection probability after one- and four-years of storage. We found that frozen and ethanol-preserved filters had significantly higher DNA concentrations and detection rates than samples preserved in Longmire's lysis buffer when DNA extraction methods differed among the treatment groups. Substantial inhibition was observed in the Longmire's samples when using a phenol-chloroform-isoamyl alcohol (PCI) extraction method, but did not occur when Longmire's samples were extracted using a Qiagen DNeasy Blood & Tissue Kit. PCI extraction also caused reduced yield and detection rates in ethanol-preserved samples, demonstrating its relative inefficiency for eDNA recovery. eDNA yield and detection rates were highly stable over one- and four-years of storage for all preservation strategies except for ethanol samples stored at room temperature, in which concentrations, but not detection rates, declined significantly after 4 years. Overall, frozen and Longmire's preserved samples had higher yield than ethanol-preserved samples, although detections were high across all media. Our study contributes valuable information towards the optimisation and standardisation of long-term storage protocols of filtered eDNA samples.