ABSTRACT Advances in museomics have expanded from dry and skeletal specimens to now include fluid‐preserved material as a source of DNA. However, our understanding of patterns of DNA degradation across specimens and tissue types remains limited, hindering justification for targeting optimal DNA sources from museum voucher specimens. Here we quantify DNA quality across taxonomic groups (amphibians, squamates, and birds) and tissue types to benchmark DNA yield, fragmentation, endogenous content, and DNA damage in fluid‐preserved specimens versus more commonly used sources (frozen tissues, dried skins, and skeletons). Our tests showed that fluid‐preserved material had smaller fragments, decreased endogenous content, and increased deamination with age, indicative of extensive degradation over even short timescales. Unexpectedly, formalin‐fixed fluid‐preserved specimens did not show clear declines in DNA yield or fragment length with specimen age, suggesting formalin fixation initially fragments DNA but may partly decouple subsequent fragment‐length patterns from specimen age. DNA yield, fragment size, and endogenous content across tissue types in fluid‐preserved specimens showed both time‐ and tissue‐type dependent patterns. DNA from liver had the highest quality metrics in younger specimens but performed worse in the oldest specimens. In older specimens, the optimal tissue types included skin, muscle or bone across taxonomic groups. However, substantial variation between individual specimens of the same preparation type and age indicated that collection history also influences DNA degradation. Our comparative analyses reinforce earlier findings that fluid‐preserved specimens of all ages are viable DNA sources. We use these findings to refine DNA sampling strategies for incorporating fluid‐preserved voucher specimens into museomic research.
ABSTRACT Documenting accelerating global biodiversity decline requires monitoring tools that can operate across large spatial scales and repeated timeframes. Analysis of airborne environmental DNA (eDNA) is an emerging method used to track species distributions, but it is unknown whether airborne eDNA retains coherent ecological signals across terrestrial landscapes, or whether atmospheric mixing and DNA transport make it too spatially diffuse to resolve meaningful macroecological patterns. To address this question, we conducted a biodiversity survey using car‐mounted airborne eDNA sampling along a repeated 1155 km route across south‐western Western Australia, spanning the Southwest Australian biodiversity hotspot and adjacent inland regions. This extensive and environmentally heterogeneous region faces ongoing pressures from habitat loss, invasive species and climate change, highlighting the need for scalable biodiversity monitoring approaches. Samples were collected at ~26 km intervals and analysed using metabarcoding assays targeting plants, vertebrates and arthropods, with generalised dissimilarity modelling used to assess environmental drivers of community turnover. We detected a broad range of plants and animals, including species of ecological and biosecurity importance. Different taxonomic groups showed distinct spatial scales of airborne eDNA community similarity and beta diversity was structured primarily by habitat and environmental gradients rather than geographic distance alone. This indicates that airborne eDNA did not simply reflect spatially homogenised biological material but retained ecological structure informative of macroecological patterns. Together, these findings demonstrate that car‐mounted airborne eDNA can recover ecologically meaningful patterns of community turnover across heterogeneous terrestrial landscapes and support landscape‐scale biodiversity assessment.
ABSTRACT One key challenge in characterizing plant communities is determining the relative abundance of distinct species in root biomass. Metabarcoding is increasingly used to estimate a species' proportion in root mass based on its proportion in sample reads, but results have been mixed in accurately converting sequencing output to species mass in mixture. We created calibration mixtures from four focal plant species to determine how the association between a species' proportion in mass and its proportion in sequencing reads of the rbcL chloroplast gene varied across sample years, soil depth, and non‐target proportion of reads. We also determined whether including these explanatory variables in prediction models could improve accuracy. We found the association between a species' proportion in reads and its proportion in mass varied across years and non‐target proportion of reads, and to a lesser extent, rooting depth, and incorporating these explanatory factors into prediction models improved prediction accuracy. A secondary objective was to determine the number of calibration levels and sequencing depth required to accurately predict a species' proportion in mass. Through simulations, we estimated that 42 calibration samples (seven proportional levels * three years * two depths) and 700 reads per sample consistently obtained accurate predictions for simple mixtures. The association between a species' proportion in mass and proportion in reads can vary considerably, and our study suggests that measuring and explaining this variation can lead to more accurate predictions of the relative abundance of plants within root biomass using metabarcoding.
ABSTRACT Sampling environmental nucleic acids (eDNA and eRNA, collectively eNA) in aquatic systems facilitates the efficient exploration of the distribution of aquatic species. Since eRNA is inherently less stable than eDNA, eRNA may yield more spatiotemporally precise information about organisms, but this volatility also poses unique methodological challenges, making it unclear whether eDNA protocols are suitable for eRNA samples. To address this gap, we conducted experiments using naturally occurring eRNA and eDNA from the mitochondrial cytochrome b (CytB) gene from largemouth bass ( Micropterus salmoides ) along with DNA and RNA extracted from bluegill tissue ( Lepomis megalotis ). We first conducted a degradation experiment to test the efficacy of refrigeration for eRNA preservation (specifically mitochondrial messenger eRNA, emRNA), given its widespread use for eDNA samples. We then compared a suite of approaches for the long‐term preservation and co‐extraction of emRNA and eDNA in the laboratory. Despite the potential that the instability of eRNA might render eDNA approaches unsuitable, we found that methods developed for eDNA samples can be readily repurposed for emRNA. Contrary to our hypothesis that refrigeration of water samples might not efficiently preserve emRNA in the short term, samples in a cooler on ice showed no decrease in concentration or detectability over 25 h for largemouth bass (Tukey's, all p > 0.3). Filtration time (i.e., time spent at room temperature) also slightly reduced eRNA yields, with concentration decreasing as filtration time increased for largemouth bass emRNA (LM, p = 0.05), highlighting the importance of cold conditions throughout processing. Additionally, as we hypothesized, a low‐cost chloroform extraction protocol yielded equivalent emRNA quantities to more expensive commercial kits for both largemouth bass and bluegill (Tukey's, p between methods > 0.8), in some cases even yielding higher concentrations than these methods. Overall, these results demonstrate the possible practical feasibility of eRNA sampling and provide guidance for future work on eRNA in natural systems.
ABSTRACT Natural history and agricultural collections contain hundreds of millions of specimens classified in terms of time, space and taxonomy. Since the first success of ancient DNA (aDNA) isolation in the 1980s, these repositories, including herbaria for plants, have been intensively used to support studies in taxonomy, macroevolution and genetic responses to anthropogenic activities. For herbarium specimens, aDNA is usually extracted from leaf samples and is highly fragmented (typically length of 50–100 bp). To optimise the amount of data retrieved and minimise destructive sampling, we isolated DNA from an unconventional plant tissue type—seed embryos. We carried out whole‐genome sequencing and compared sequenced DNA quality between embryo and leaf tissue from the same specimen. We evaluated endogenous DNA proportion, median fragment length, damage fraction per site ( λ ), decay rates ( k ), nucleotide misincorporations and library complexity for three species: cultivated rice Oryza sativa , wild rice O. rufipogon and wild barley Hordeum spontaneum . In O. sativa , embryos exhibited significantly higher endogenous content and median fragment length than leaves. The superior DNA preservation was likely due to the protective role of the seed husk, which might play an important role in DNA preservation in plants collected in the tropics. By contrast, in temperate H. spontaneum , tissue type had minimal impact on DNA quality. Despite the minuscule size of the embryos, all derived genomic libraries were highly complex, sufficient for deep whole genome sequencing. These results highlight seed embryos as a promising alternative aDNA source for herbarium specimens, economic botany and anthropological museum collections.
