Molecular Ecology Resources最新文献

Understanding diet composition is essential for unravelling trophic interactions in aquatic ecosystems. DNA metabarcoding, utilising various variable regions of the 18S rRNA gene, is increasingly employed to investigate zooplankton diet composition. However, accurate results depend on rapid inactivation of digestive enzymes and DNA nucleases through proper sample processing and preservation. In this study, we compare the prey communities of Antarctic krill retrieved from the 18S variable regions V4 and V7 and assess how different processing treatments affect the detected prey composition of both krill and salps. Our findings highlight the critical importance of prompt sample processing for species with highly efficient digestive enzymes, such as krill, to preserve rapidly digested prey, including gelatinous plankton. Comparative analyses of the V4 and V7 regions revealed significantly different prey communities within the same krill samples, indicating that these regions may not be suitable for direct comparisons within or across studies. To complement molecular approaches, we also analyse fatty acids (FA) as trophic markers which provide insights into dietary habits over both short and long time scales. By comparing FA signals from stomach and tissue samples of the same krill and salp individuals, we identified significant differences in trophic markers representing different plankton groups. These findings emphasise the necessity of separating digestive tract from tissue to distinguish between short- and long-term diet signals. Furthermore, integrating FA analysis with metabarcoding offers valuable insights into zooplankton digestion efficiency across taxonomic levels. This combined approach enhances our understanding of zooplankton feeding ecology and trophic interactions in marine ecosystems.

Efficient use of environmental nucleic acids (eNAs) in freshwater biodiversity monitoring requires understanding their degradation and detectability in interconnected ecosystems. We employed a novel field-scale assay to compare environmental DNA (eDNA) and environmental RNA (eRNA) decay rates and detectability across four genetic markers (16S, 18S, COI and LDHA) in connected and isolated 1000-L mesocosms containing natural planktonic assemblages. This design provides ecologically relevant and complex settings to assess how connectivity influences the detectability of eNA over time. Isolated and head mesocosms were spiked with eNAs from cultured Daphnia pulex, absent from the water source, while downstream mesocosms received eNAs via unidirectional water transfers. Using digital PCR (dPCR), we captured fine-scale temporal patterns across mitochondrial and nuclear markers and transcript types (mRNA and rRNA), an approach rarely combined in previous research. eRNA degraded significantly faster than eDNA across markers and mesocosm types. Among RNA types, mRNA (COI, LDHA) degraded faster than rRNA (16S, 18S). eRNA followed a uniform monophasic decay pattern, whereas eDNA displayed biphasic decay for nuclear markers and monophasic decay for mitochondrial markers. eNA decay rates in this field-relevant mesocosm network exceeded those from laboratory scale. While decay rates remained consistent across networks, detectability declined with dilution. Even after a 10,000-fold dilution, both eNAs were detected in terminal mesocosms, demonstrating effective transport across the network. Although RNA degrades rapidly, high detectability was achieved across diverse dilutions using dPCR, highlighting eRNA's potential for detecting active biological communities in freshwater systems.

Genotype imputation, the inference of missing genotypes using a reference set of population haplotypes, is a cost-effective tool for improving the quality and quantity of genetic datasets. Imputation is usually applied to large and well-characterised datasets of humans and livestock, even though it could also benefit smaller natural populations. This study aims to understand the best practices and effectiveness of imputation with a small reference panel for species with low genetic diversity, using a case study of a population of the hihi/stitchbird (Notiomystis cincta). We used a leave-one-out method to test imputation on 30 high-coverage hihi individuals where SNPs were masked before being imputed with Beagle v5.4. Imputation accuracy was measured using r², the correlation between imputed and ground truth genotype dosages. We tested combinations of five imputation parameters, the inclusion of two linkage maps, reference panels of different sizes and compositions and targets of various SNP densities and sporadic missingness. We achieved mean r² exceeding 0.95 in most tests from a small reference panel of high-fecundity individuals. Imputation accuracy was not improved by including a linkage map and decreased at very low SNP densities. Imputed SNPs were filtered using r² to assess downstream heterozygosity calculations, the site frequency spectrum (SFS) and inference of runs of homozygosity (ROHs). We found that filtering and SNP density greatly affected heterozygosity and SFS at low SNP densities but that ROH inference was relatively robust to both. We provide a template for testing and optimising imputation in other wild populations.

DNA barcoding traditionally relies on Sanger sequencing but faces limitations with degraded samples. High-throughput sequencing (HTS) offers a cost-effective alternative, enabling rapid barcode generation for extensive datasets. The advantage of HTS is its ability to employ multiplexing strategies, allowing thousands of samples to be processed simultaneously in a single sequencing run. This study presents the CODEX approach, a double-indexed HTS method designed to sequence overlapping cox-1 barcode fragments, suitable for samples with degraded DNA. The approach was applied to neogastropods, a diverse lineage of marine molluscs, using specimens (both recently collected and relatively older) from the Muséum national d'Histoire naturelle (MNHN) collections. The pipeline was used to process 15,076 samples, yielding 10,905 cleaned and assembled sequences, achieving a success rate of 72.33%. The CODEX method demonstrated advantages over Sanger sequencing by enabling the recovery of barcodes from samples previously deemed unsuitable, with significantly reduced costs (€0.5 per sequence vs. €4.5). Notably, DNA quality and sequencing success were strongly correlated with collection date, emphasising the impact of preservation methods and storage conditions. Sequencing success rates varied among families but were not correlated with phylogenetic relationships or specimen size, indicating the robustness of the primers designed for neogastropods. This study highlights the efficiency of the CODEX approach for large-scale DNA barcoding projects, especially when handling degraded samples. The CODEX pipeline and associated resources are publicly accessible, offering a scalable solution for molecular systematics and beyond.

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.

DNA methylation is crucial for genome regulation and provides key insights into the interaction between genetics and environmental factors, offering valuable perspectives for ecological research. However, knowledge of DNA methylation patterns in nonmodel invertebrates remains limited. The present study addresses this knowledge gap by conducting the first methylome profiling of the Pacific crown-of-thorns seastar (CoTS; Acanthaster cf. solaris), a coral-eating species that aggravates the decline of Indo-Pacific coral reefs. Using Oxford Nanopore Technology (ONT) we generated long-read sequences, covering over 90% of CpG dinucleotides in the CoTS genome. Our analysis revealed a mosaic methylation landscape with moderate genome-wide methylation levels of 37.7%. Comparative analysis highlights the intermediate methylation state observed in other deuterostome invertebrates, positioning them between the hypomethylated genomes of protostomes and the hypermethylated genomes of vertebrates. Methylation in CoTS was predominantly localised within gene bodies, especially in intronic regions, enabling modulation of gene expression and potentially supporting fitness in dynamic marine environments. Additionally, elevated methylation in repetitive elements suggests a role in genome defence. This study demonstrates the effectiveness of ONT for comprehensive methylome analysis in ecologically important nonmodel species and deepens our understanding of the epigenetic landscape in deuterostome invertebrates. We also present a detailed laboratory and bioinformatics workflow, including modified phenol–chloroform protocols that address the challenge of extracting high molecular weight DNA from marine invertebrates. Together with the methylome profiles, these resources serve as a foundation for future research, enabling investigations into DNA methylation functions, applications for CoTS outbreak management and comparative studies across diverse lineages.

Assessing and monitoring genetic diversity is vital for understanding the ecology and evolution of natural populations but is often challenging in animal and plant species due to technically and physically demanding tissue sampling. Although environmental DNA (eDNA) metabarcoding is a promising alternative to the traditional population genetic monitoring based on biological samples, its practical application remains challenging due to spurious sequences present in the amplicon data, even after data processing with the existing sequence filtering and denoising (error correction) methods. Here we developed a novel amplicon filtering approach that can effectively eliminate such spurious amplicon sequence variants (ASVs) in eDNA metabarcoding data. A simple simulation of eDNA metabarcoding processes was performed to understand the patterns of read count (abundance) distributions of true ASVs and their polymerase chain reaction (PCR)-generated artefacts (i.e., false-positive ASVs). Based on the simulation results, the approach was developed to estimate the abundance distributions of true and false-positive ASVs using Gaussian mixture models and to determine a statistically based threshold between them. The developed approach was implemented as an R package, gmmDenoise and evaluated using single-species metabarcoding datasets in which all or some true ASVs (i.e., haplotypes) were known. Example analyses using community (multi-species) metabarcoding datasets were also performed to demonstrate how gmmDenoise can be used to derive reliable intraspecific diversity estimates and population genetic inferences from noisy amplicon sequencing data. The gmmDenoise package is freely available in the GitHub repository (https://github.com/YSKoseki/gmmDenoise).