Molecular Ecology Resources最新文献

Amphibians are the most threatened group of vertebrates and are in dire need of conservation intervention to ensure their continued survival. They exhibit unique features including a high diversity of reproductive strategies, permeable and specialized skin capable of producing toxins and antimicrobial compounds, multiple genetic mechanisms of sex determination and in some lineages, the ability to regenerate limbs and organs. Although genomic approaches would shed light on these unique traits and aid conservation, sequencing and assembly of amphibian genomes has lagged behind other taxa due to their comparatively large genome sizes. Fortunately, the development of long-read sequencing technologies and initiatives has led to a recent burst of new amphibian genome assemblies. Although growing, the field of amphibian genomics suffers from the lack of annotation resources, tools for working with challenging genomes and lack of high-quality assemblies in multiple clades of amphibians. Here, we analyse 51 publicly available amphibian genomes to evaluate their usefulness for functional genomics research. We report considerable variation in genome assembly quality and completeness and report some of the highest transposable element and repeat contents of any vertebrate. Additionally, we detected an association between transposable element content and climatic variables. Our analysis provides evidence of conserved genome synteny despite the long divergence times of this group, but we also highlight inconsistencies in chromosome naming and orientation across genome assemblies. We discuss sequencing gaps in the phylogeny and suggest key targets for future sequencing endeavours. Finally, we propose increased investment in amphibian genomics research to promote their conservation.

Microhaplotypes are small linked genomic regions comprising two or more single-nucleotide polymorphisms (SNPs) that are being applied in forensics and are emerging in wildlife monitoring studies and genomic epidemiology. Typically, targeted in non-coding regions, microhaplotypes in exonic regions can be designed with larger amplicons to capture functional non-synonymous sites and minimise insertion/deletion (indel) polymorphisms. Quality control is an important first step for high-confidence genotyping to counteract such false-positive variants. As genetic markers with higher polymorphism compared to biallelic SNPs, it is critical to ensure sequencing errors across the microhaplotype amplicon are filtered out to avoid introducing false-haplotypes. We developed the MhGeneS pipeline which works in tandem with Seq2Sat to help validate microhaplotype genotyping of the coding region of genes, with broader applicability to any microhaplotype profiling. We genotyped microhaplotype regions of the Zfx (≅ 160 bp) and Zfy (≅ 140 bp) genes, as well as an exon of the prion protein (Prnp) gene (≅ 370 bp) in caribou (Rangifer tarandus) using paired-end Illumina technology. As important quality metrics affecting microhaplotype calling, we identified the sequencing error rate profile related to the overlap or non-overlap of paired-end reads as well as the read depth as significant. In the case of Prnp, we achieved confident microhaplotype calling through MhGeneS by removing small sections of the 5′ and 3′ amplicons and using a minimum read depth of 20. Read depth and sequence trimming may be locus-specific, and validation of these parameters is recommended before the high-throughput profiling of samples.

Mitigating ongoing losses of insects and their key functions (e.g. pollination) requires tracking large-scale and long-term community changes. However, doing so has been hindered by the high diversity of insect species that requires prohibitively high investments of time, funding and taxonomic expertise when addressed with conventional tools. Here, we show that these concerns can be addressed through a comprehensive, scalable and cost-efficient DNA metabarcoding workflow. We use 1815 samples from 75 Malaise traps across Germany from 2019 and 2020 to demonstrate how metabarcoding can be incorporated into large-scale insect monitoring networks for less than 50 € per sample, including supplies, labour and maintenance. We validated the detected species using two publicly available databases (GBOL and GBIF) and the judgement of taxonomic experts. With an average of 1.4 M sequence reads per sample we uncovered 10,803 validated insect species, of which 83.9% were represented by a single Operational Taxonomic Unit (OTU). We estimated another 21,043 plausible species, which we argue either lack a reference barcode or are undescribed. The total of 31,846 species is similar to the number of insect species known for Germany (~35,500). Because Malaise traps capture only a subset of insects, our approach identified many species likely unknown from Germany or new to science. Our reproducible workflow (~80% OTU-similarity among years) provides a blueprint for large-scale biodiversity monitoring of insects and other biodiversity components in near real time.

Snake venoms are complex mixtures of toxic proteins that hold significant medical, pharmacological and evolutionary interest. To better understand the genetic diversity underlying snake venoms, we developed VenomCap, a novel exon-capture probe set targeting toxin-coding genes from a wide range of elapid snakes, with a particular focus on the ecologically diverse and medically important subfamily Hydrophiinae. We tested the capture success of VenomCap across 24 species, representing all major elapid lineages. We included snake phylogenomic probes in the VenomCap capture set, allowing us to compare capture performance between venom and phylogenomic loci and to infer elapid phylogenetic relationships. We demonstrated VenomCap's ability to recover exons from ~1500 target markers, representing a total of 24 known venom gene families, which includes the dominant gene families found in elapid venoms. We find that VenomCap's capture results are robust across all elapids sampled, and especially among hydrophiines, with respect to measures of target capture success (target loci matched, sensitivity, specificity and missing data). As a cost-effective and efficient alternative to full genome sequencing, VenomCap can dramatically accelerate the sequencing and analysis of venom gene families. Overall, our tool offers a model for genomic studies on snake venom gene diversity and evolution that can be expanded for comprehensive comparisons across the other families of venomous snakes.

For two decades, DNA barcoding and, more recently, DNA metabarcoding have been used for molecular species identification and estimating biodiversity. Despite their growing use, few studies have systematically evaluated these methods. This study aims to evaluate the efficacy of barcoding methods in identifying species and estimating biodiversity, by assessing their consistency with traditional morphological identification and evaluating how assignment consistency is influenced by taxonomic group, sequence similarity thresholds and geographic distance. We first analysed 951 insect specimens across three taxonomic groups: butterflies, bumblebees and parasitic wasps, using both morphological taxonomy and single-specimen COI DNA barcoding. An additional 25,047 butterfly specimens were identified by COI DNA metabarcoding. Finally, we performed a systematic review of 99 studies to assess average consistency between insect species identity assigned via morphology and COI barcoding and to examine the distribution of research effort. Species assignment consistency was influenced by taxonomic group, sequence similarity thresholds and geographic distance. An average assignment consistency of 49% was found across taxonomic groups, with parasitic wasps displaying lower consistency due to taxonomic impediment. The number of missing matches doubled with a 100% sequence similarity threshold and COI intraspecific variation increased with geographic distance. Metabarcoding results aligned well with morphological biodiversity estimates and a strong positive correlation between sequence reads and species abundance was found. The systematic review revealed an 89% average consistency and also indicated taxonomic and geographic biases in research effort. Together, our findings demonstrate that while problems persist, barcoding approaches offer robust alternatives to traditional taxonomy for biodiversity assessment.

DNA methylation (DNAm) is a mechanism for rapid acclimation to environmental conditions. In natural systems, small effect sizes relative to noise necessitates large sampling efforts to detect differences. Large numbers of individually sequenced libraries are costly. Pooling DNA prior to library preparation may be an efficient way to reduce costs and increase sample size, yet there are to date no recommendations in ecological epigenetics research. We test whether pooled and individual libraries yield comparable DNAm signals in a natural system exposed to different pollution levels by generating whole-epigenome data from two invasive molluscs (Corbicula fluminea, Dreissena polymorpha) collected from polluted and unpolluted localities (Italy). DNA of the same individuals were used for pooled and individual epigenomic libraries and sequenced with equivalent resources per individual. We found that pooling effectively captures similar genome-wide and global methylation signals as individual libraries, highlighting that pooled libraries are representative of the global population signal. However, pooled libraries yielded orders of magnitude more data than individual libraries, which was a consequence of higher coverage. We would therefore recommend aiming for a high initial coverage of individual libraries (15×) in future studies. Consequently, we detected many more differentially methylated regions (DMRs) with the pooled libraries and a significantly lower statistical power for regions from individual libraries. Computationally pooled data from the individual libraries produced fewer DMRs and the overlap with wet-lab pooled DMRs was relatively low. We discuss possible causes for discrepancies, list benefits and drawbacks of pooling, and provide recommendations for future epigenomic studies.

Antarctic krill (Euphausia superba Dana) is a keystone species in the Southern Ocean ecosystem, with ecological and commercial significance. However, its vulnerability to climate change requires an urgent investigation of its adaptive potential to future environmental conditions. Historical museum collections of krill from the early 20th century represent an ideal opportunity to investigate how krill have changed over time due to predation, fishing and climate change. However, there is currently no cost-effective method for implementing population scale collection genomics for krill given its genome size (48 Gbp). Here, we assessed the utility of two inexpensive methods for population genetics using historical krill samples, specifically low-coverage shotgun sequencing (i.e. ‘genome-skimming’) and exome capture. Two full-length transcriptomes were generated and used to identify 166 putative gene targets for exome capture bait design. A total of 20 historical krill samples were sequenced using shotgun and exome capture. Mitochondrial and nuclear ribosomal sequences were assembled from both low-coverage shotgun and off-target of exome capture data demonstrating that endogenous DNA sequences could be assembled from historical collections. Although, mitochondrial and ribosomal sequences are variable across individuals from different populations, phylogenetic analysis does not identify any population structure. We find exome capture provides approximately 4500-fold enrichment of sequencing targeted genes, suggesting this approach can generate the sequencing depth required to call identify a significant number of variants. Unlocking historical collections for genomic analyses using exome capture, will provide valuable insights into past and present biodiversity, resilience and adaptability of krill populations to climate change.

The origin of sociality represents one of the most important evolutionary transitions. Insect sociality evolved in some hemipteran aphids, which can produce soldiers and normal nymphs with distinct morphology and behaviour through parthenogenesis. The lack of genomic data resources has hindered the investigations into molecular mechanisms underlying their social evolution. Herein, we generated the first chromosomal-level genome of a social hemipteran (Pseudoregma bambucicola) with highly specialized soldiers and performed comparative genomic and transcriptomic analyses to elucidate the molecular signatures and regulatory mechanisms of caste differentiation. P. bambucicola has a larger known aphid genome of 582.2 Mb with an N50 length of 11.24 Mb, and about 99.6% of the assembly was anchored to six chromosomes with a scaffold N50 of 98.27 Mb. A total of 14,027 protein-coding genes were predicted and 37.33% of the assembly were identified as repeat sequences. The social evolution is accompanied by a variety of changes in genome organization, including expansion of gene families related to transcription factors, transposable elements, as well as species-specific expansions of certain sugar transporters and UGPases involved in carbohydrate metabolism. We also characterized large candidate gene sets linked to caste differentiation and found evidence of expression regulation and positive selection acting on energy metabolism and muscle structure, explaining the soldier-specific traits including morphological and behavioural specialization, developmental arrest and infertility. Overall, this study offers new insights into the molecular basis of social aphids and the evolution of insect sociality and also provides valuable data resources for further comparative and functional studies.

Fish ear bones, known as otoliths, are often collected in fisheries to assist in management, and are a common sample type in museum and national archives. Beyond their utility for ageing, morphological and trace element analysis, otoliths are a repository of valuable genomic information. Previous work has shown that DNA can be extracted from the trace quantities of tissue remaining on the surface of otoliths, despite the fact that they are often stored dry at room temperature. However, much of this work has used reduced representation sequencing methods in clean lab conditions, to achieve adequate yields of DNA, libraries and ultimately single-nucleotide polymorphisms (SNPs). Here, we pioneer the use of small-scale (spike-in) sequencing to screen contemporary otolith samples prepared in regular molecular biology (in contrast to clean) laboratories for contamination and quality levels, submitting for whole-genome resequencing only samples above a defined endogenous DNA threshold. Despite the typically low quality and quantity of DNA extracted from otoliths, we are able to produce whole-genome libraries and ultimately sets of filtered, unlinked and even putatively adaptive SNPs of ample numbers for downstream uses in population, climate and conservation genomics. By comparing with a set of tissue samples from the same species, we are able to highlight the quality and efficacy of otolith samples from DNA extraction and library preparation, to bioinformatic preprocessing and SNP calling. We provide detailed schematics, protocols and scripts of our approach, such that it can be adopted widely by the community, improving the use of otoliths as a source of valuable genomic data.