Molecular Ecology Resources最新文献

Fungi play a vital role in ecosystem functioning, yet significant knowledge gaps persist in understanding their diversity and distribution leading to uncertainties about their threat status and extinction risk. This is partly owed to the difficulty of monitoring fungi using traditional fruiting body surveys. The present study evaluates airborne environmental DNA (eDNA) sampling as a monitoring tool with a focus on grassland macrofungi. We applied active and passive air sampling methods, complemented by extensive field surveys of waxcap and clavarioid fungi–species groups of high relevance for conservation. Twenty-nine species were recorded during the field surveys, 19 of which were also detectable by ITS2 metabarcoding of the collected samples. An additional 12 species from the studied genera were identified exclusively in air eDNA. We found that the patterns of species detection and read abundance in air samples reflected the abundance and occurrence of fruiting bodies on the field. Dispersal kernels fitted for the three dominant species predicted rapidly decreasing spore concentrations with increasing distance from fruitbodies. Airborne assemblages were dominated by a high diversity of common species, while rare and threatened red-listed species were under-represented, which underscores the difficulty in detecting rare species, not only in conventional surveys. Considering the benefits and drawbacks of air sampling and fruitbody surveys, we conclude that air sampling serves as a cost- and time-efficient tool to characterize local macrofungal communities, providing the potential to facilitate and improve future fungal monitoring efforts.

The mapmixture R package and interactive web app are tools to aid visualisation of admixture and population structure in geographic space. The purpose of mapmixture is to enable and encourage molecular ecologists, and in particular population geneticists and phylogeneticists, to plot their admixture, ancestry or assignment results on a map when location information is available. mapmixture accepts data in the format typically generated by admixture analyses and visualises proportions to each genetic cluster per site as pie charts on a projected (optional) map. Combining this site-based map presentation approach with the routine individual-based presentation of admixture (structure) barplots will enhance interpretation of genetic-geographic patterns. Additionally, in the context of science communication, this enables clearer transfer of spatial genetic information to readers or listeners, and especially to audiences that do not have a background in genetics but who are able to use the genetic information as evidence in conservation management. The latest version of mapmixture is available on GitHub (https://github.com/tom-jenkins/mapmixture), which details installation instructions and examples of how to use the package and interactive web app.

Age is a key demographic in conservation where age classes show differences in important population metrics such as morbidity and mortality. Several traits, including reproductive potential, also show senescence with ageing. Thus, the ability to estimate age of individuals in a population is critical in understanding the current structure as well as their future fitness. Many methods exist to determine age in wildlife, with most using morphological features that show inherent variability with age. These methods require significant expertise and become less accurate in adult age classes, often the most critical groups to model. Molecular methods have been applied to measuring key population attributes, and more recently epigenetic attributes such as methylation have been explored as biomarkers for age. There are, however, several factors such as permits, sample sovereignty, and costs that may preclude the use of extant methods in a conservation context. This study explored the utility of measuring age-related changes in methylation in candidate genes using mass array technology. Novel methods are described for using gene orthologues to identify and assay regions for differential methylation. To illustrate the potential application, African cheetah was used as a case study. Correlation analyses identified six methylation sites with an age relationship, used to develop a model with sufficient predictive power for most conservation contexts. This model was more accurate than previous attempts using PCR and performed similarly to candidate gene studies in other mammal species. Mass array presents an accurate and cost-effective method for age estimation in wildlife of conservation concern.

Environmental DNA (eDNA) is used for biodiversity assessments in a variety of ecosystems across the globe, whereby different eDNA concentration, preservation and extraction methods can outperform others depending on the sampling conditions and environment. Tropical and subtropical ecosystems in Africa are among the less studied systems concerning eDNA-based monitoring. Waterholes in arid parts of southern Africa represent important agglomeration points for terrestrial mammals, and the eDNA shed into such waterbodies provides a powerful source of information for monitoring mammalian biodiversity in the surrounding area. However, the applied methods for eDNA sampling, preservation and filtering in different freshwater systems vary greatly, and rigorous protocol testing in African freshwater systems is still lacking. This study represents the first attempt to examine variations in eDNA concentration, preservation and extraction methods under remote field conditions using waterborne eDNA in a savanna system. Collected samples were heavily affected by microalgal and bacterial growth, impeding eDNA capture and PCR success. We demonstrate clear effects of the methodological choices, which also depend on the state of eDNA. A preliminary metabarcoding run showed little taxonomic overlap in mammal species detection between two metabarcoding primers tested. We recommend water filtering (using filters with pore sizes >1 μm) over centrifugation for eDNA concentration, Longmire's solution for ambient temperature sample preservation and Qiagen's DNeasy PowerSoil Pro Kit for DNA extraction of these inhibitor-prone samples. Furthermore, at least two independent metabarcoding markers should be utilized in order to maximize species detections in metabarcoding studies.

Utilization of faeces has long been a popular approach for genetic and ecological studies of wildlife. However, the success of molecular marker genotyping and genome resequencing is often unpredictable due to insufficient enrichment of endogenous DNA in the total faecal DNA that is dominated by bacterial DNA. Here, we report a simple and cheap method named PEERS to predominantly lyse animal cells over bacteria by using sodium dodecyl sulphate so as to discharge endogenous DNA into liquid phase before bacterial DNA. By brief centrifugation, total DNA with enriched endogenous fraction can be extracted from the supernatant using routine methods. Our assessments showed that the endogenous DNA extracted by PEERS was significantly enriched for various types of faeces from different species, preservation time and conditions. It significantly improves the genotyping correctness and efficiency of genome resequencing with the total additional cost of $ 0.1 and a short incubation step to treat a faecal sample. We also provide methods to assess the enrichment efficiency of mitochondrial and nuclear DNA and models to predict the usability of faecal DNA for genotyping of short tandem repeat, single-nucleotide polymorphism and whole-genome resequencing.

As the scope of plant eDNA metabarcoding diversifies, so do the primers, markers and methods. A wealth of primers exists today, but their comparative evaluation is lacking behind. Similarly, multi-marker approaches are recommended but debates persist regarding barcode complementarity and optimal combinations. After a literature compilation of used primers, we compared in silico 102 primer pairs based on amplicon size, coverage and specificity, followed by an experimental evaluation of 15 primer pairs on a mock community sample covering 268 plant species and genera, and about 100 families. The analysis was done for the four most common plant metabarcoding markers, rbcL, trnL, ITS1 and ITS2 and their complementarity was assessed based on retrieved species. By focusing on existing primers, we identify common designs, promote alternatives and enhance prior-supported primers for immediate applications. The ITS2 was the best-performing marker for flowering vascular plants and was congruent to ITS1. However, the combined taxonomic breadth of ITS2 and rbcL surpassed any other combination, highlighting their high complementarity across Streptophyta. Overall, our study underscores the significance of comprehensive primer and barcode evaluations tailored to metabarcoding applications.

Surveying biodiversity has taken a quantum leap with environmental DNA (eDNA) metabarcoding, an immensely powerful approach lauded for its efficiency, sensitivity, and non-invasiveness. This approach emerges as a game-changer for the elusive realm of endangered and rare species—think nocturnal, environmentally elusive amphibians. Here, we have established a framework for constructing a reliable metabarcoding pipeline for amphibians, covering primer design, performance evaluation, laboratory validation, and field validation processes. The Am250 primer, located on the mitochondrial 16S gene, was optimal for the eDNA monitoring of amphibians, which demonstrated higher taxonomic resolution, smaller species amplification bias, and more extraordinary detection ability compared to the other primers tested. Am250 primer exhibit an 83.8% species amplification rate and 75.4% accurate species identification rate for Chinese amphibians in the in silico PCR and successfully amplified all tested species of the standard samples in the in vitro assay. Furthermore, the field-based mesocosm experiment showed that DNA can still be detected by metabarcoding even days to weeks after organisms have been removed from the mesocosm. Moreover, field mesocosm findings indicate that eDNA metabarcoding primers exhibit different read abundances, which can affect the relative biomass of species. Thus, appropriate primers should be screened and evaluated by three experimental approaches: in silico PCR simulation, target DNA amplification, and mesocosm eDNA validation. The selection of a single primer set or multiple primers' combination should be based on the monitoring groups to improve the species detection rate and the credibility of results.

Using high-throughput sequencing for precise genotyping of multi-locus gene families, such as the major histocompatibility complex (MHC), remains challenging, due to the complexity of the data and difficulties in distinguishing genuine from erroneous variants. Several dedicated genotyping pipelines for data from high-throughput sequencing, such as next-generation sequencing (NGS), have been developed to tackle the ensuing risk of artificially inflated diversity. Here, we thoroughly assess three such multi-locus genotyping pipelines for NGS data, the DOC method, AmpliSAS and ACACIA, using MHC class IIβ data sets of three-spined stickleback gDNA, cDNA and “artificial” plasmid samples with known allelic diversity. We show that genotyping of gDNA and plasmid samples at optimal pipeline parameters was highly accurate and reproducible across methods. However, for cDNA data, the gDNA-optimal parameter configuration yielded decreased overall genotyping precision and consistency between pipelines. Further adjustments of key clustering parameters were required tο account for higher error rates and larger variation in sequencing depth per allele, highlighting the importance of template-specific pipeline optimization for reliable genotyping of multi-locus gene families. Through accurate paired gDNA-cDNA typing and MHC-II haplotype inference, we show that MHC-II allele-specific expression levels correlate negatively with allele number across haplotypes. Lastly, sibship-assisted cDNA-typing of MHC-I revealed novel variants linked in haplotype blocks, and a higher-than-previously-reported individual MHC-I allelic diversity. In conclusion, we provide novel genotyping protocols for the three-spined stickleback MHC-I and -II genes, and evaluate the performance of popular NGS-genotyping pipelines. We also show that fine-tuned genotyping of paired gDNA-cDNA samples facilitates amplification bias-corrected MHC allele expression analysis.

Environmental DNA (eDNA) is an effective tool for describing fish biodiversity in lotic environments, but the downstream transport of eDNA released by organisms makes it difficult to interpret species detection at the local scale. In addition to biophysical degradation and exchanges at the water–sediment interface, hydrological conditions control the transport distance. A new eDNA transport model described in this paper considers downstream retention and degradation processes in combination with hydraulic conditions and assumes that the sedimentation rate of very fine particles is a correct estimate of the eDNA deposition rate. Based on meta-analyses of available studies, the particle size distribution of fish eDNA (PSD), the relationship between the sedimentation rate and the size of very fine particles in suspension, and the influence of temperature on the degradation rate of fish eDNA were successively modelled. After combining the results in a mechanistic-based model, the eDNA uptake distances (distance required to retain 63.21% of the eDNA particles in the riverbed) observed in a compilation of previous experimental studies were correctly simulated. eDNA degradation is negligible at low flow and temperature but has a comparable influence to background transfer when hydraulic conditions allow a long uptake distance. The wide prediction intervals associated with the simulations reflect the complexity of the processes acting on eDNA after shedding. This model can be useful for estimating eDNA detection distance downstream from a source point and discussing the possibility of false positive detection in eDNA samples, as shown in an example.

Highly polymorphic markers, such as microsatellites, are invaluable for the study of natural populations. However, contemporary methods for genotyping highly polymorphic variants have serious drawbacks that impede their efficiency. We created Polly, an R package with C++ source code that uses Illumina short-read data to genotype microsatellites, detect highly polymorphic variants and identify clusters of highly polymorphic SNPs, indels and microsatellites. We tested Polly on short-read data from Xiphophorus birchmanni (Teleostei: Poeciliidae) and Arabidopsis thaliana, finding it to be efficient and accurate both for microsatellite genotyping and polymorphic marker detection. This program can be applied to any diploid population for which there exists short-read data and at least one scaffolded reference genome.