Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to detect plant and animal taxa in basic and applied biodiversity research because of its targeted nature that allows sequencing of genetic markers from many samples in parallel. To achieve this, PCR amplification is carried out with primers designed to target a taxonomically informative marker within a taxonomic group, and sample-specific nucleotide identifiers are added to the amplicons prior to sequencing. This enables assignment of the sequences back to the samples they originated from. Nucleotide identifiers can be added during the metabarcoding PCR and/or during ‘library preparation’, i.e. when amplicons are prepared for sequencing. Different strategies to achieve this labelling exist. All have advantages, challenges and limitations, some of which can lead to misleading results, and in the worst case compromise the fidelity of the metabarcoding data. Given the range of questions addressed using metabarcoding, the importance of ensuring that data generation is robust and fit for purpose should be at the forefront of practitioners seeking to employ metabarcoding for biodiversity assessments. Here, we present an overview of the three main workflows for sample-specific labelling and library preparation in metabarcoding studies on Illumina sequencing platforms. Further, we distil the key considerations for researchers seeking to select an appropriate metabarcoding strategy for their specific study. Ultimately, by gaining insights into the consequences of different metabarcoding workflows, we hope to further consolidate the power of metabarcoding as a tool to assess biodiversity across a range of applications.

We present the chromosome-level genome assembly of Dysdera silvatica Schmidt, 1981, a nocturnal ground-dwelling spider endemic from the Canary Islands. The genus Dysdera has undergone a remarkable diversification in this archipelago mostly associated with shifts in the level of trophic specialization, becoming an excellent model to study the genomic drivers of adaptive radiations. The new assembly (1.37 Gb; and scaffold N50 of 174.2 Mb), was performed using the chromosome conformation capture scaffolding technique, represents a continuity improvement of more than 4,500 times with respect to the previous version. The seven largest scaffolds or pseudochromosomes cover 87% of the total assembly size and match consistently with the seven chromosomes of the karyotype of this species, including the characteristic large X chromosome. To illustrate the value of this new resource we performed a comprehensive analysis of the two major arthropod chemoreceptor gene families (i.e., gustatory and ionotropic receptors). We identified 545 chemoreceptor sequences distributed across all pseudochromosomes, with a notable underrepresentation in the X chromosome. At least 54% of them localize in 83 genomic clusters with a significantly lower evolutionary distances between them than the average of the family, suggesting a recent origin of many of them. This chromosome-level assembly is the first high-quality genome representative of the Synspermiata clade, and just the third among spiders, representing a new valuable resource to gain insights into the structure and organization of chelicerate genomes, including the role that structural variants, repetitive elements and large gene families played in the extraordinary biology of spiders.

Arctium lappa has a long medicinal and edible history with great economic importance. We combined Illumina and PacBio sequences to generate the first high-quality chromosome-level draft genome of A. lappa. The assembled genome is approximately 1.79 Gb with a N50 contig size of 6.88 Mb. Approximately 1.70 Gb (95.4%) of the contig sequences were anchored onto 18 chromosomes using Hi-C data; the scaffold N50 was improved to be 91.64 Mb. Furthermore, we obtained 1.12 Gb (68.46%) of repetitive sequences and 32,771 protein-coding genes; 616 positively selected candidate genes were identified. Additionally, we compared the transcriptomes of A. lappa roots at three different developmental stages and identified 8,943 differentially expressed genes (DEGs) in these tissues. Among candidate genes related to lignan biosynthesis, the following were found to be highly correlated with the accumulation of arctiin: 4-coumarate-CoA ligase (4CL), dirigent protein (DIR), and hydroxycinnamoyl transferase (HCT). These data can be utilized to identify genes related to A. lappa quality or provide a basis for molecular identification and comparative genomics among related species.

Dispersal abilities play a crucial role in shaping the extent of population genetic structure, with more mobile species being panmictic over large geographic ranges and less mobile ones organized in meta-populations exchanging migrants to different degrees. In turn, population structure directly influences the coalescence pattern of the sampled lineages, but the consequences on the estimated variation of the effective population size (Ne) over time obtained by means of unstructured demographic models remain poorly understood. However, this knowledge is crucial for biologically interpreting the observed Ne trajectory and further devising conservation strategies in endangered species. Here we investigated the demographic history of four shark species (Carharhinus melanopterus, Carharhinus limbatus, Carharhinus amblyrhynchos, Galeocerdo cuvier) with different degrees of endangered status and life history traits related to dispersal distributed in the Indo-Pacific and sampled off New Caledonia. We compared several evolutionary scenarios representing both structured (meta-population) and unstructured models and then inferred the Ne variation through time. By performing extensive coalescent simulations, we provided a general framework relating the underlying population structure and the observed Ne dynamics. On this basis, we concluded that the recent decline observed in three out of the four considered species when assuming unstructured demographic models can be explained by the presence of population structure. Furthermore, we also demonstrated the limits of the inferences based on the sole site frequency spectrum and warn that statistics based on linkage disequilibrium will be needed to exclude recent demographic events affecting meta-populations.

Metabarcoding of environmental DNA (eDNA) is now widely used to build diversity profiles from DNA that has been shed by species into the environment. There is substantial interest in the expansion of eDNA approaches for improved detection of terrestrial vertebrates using invertebrate-derived DNA (iDNA) in which hematophagous, sarcophagous, and coprophagous invertebrates sample vertebrate blood, carrion, or feces. Here, we use metabarcoding and multiple iDNA samplers (carrion flies, sandflies, and mosquitos) to profile gamma and alpha diversity in a dry, tropical forest in the southern Amazon. Our main objectives were to (1) compare diversity found with iDNA to camera trapping, which is the conventional method of vertebrate diversity surveillance and (2) compare each of the iDNA samplers to assess the effectiveness, efficiency, and potential biases associated with each sampler. Carrion flies were the most effective sampler, despite the least amount of sampling effort and the fewest number of individuals captured for metabarcoding, in describing vertebrate biodiversity followed by sandflies. Camera traps had the highest median species richness at the site-level but showed strong bias towards carnivore and ungulate species and missed much of the diversity described by iDNA methods. Mosquitos showed a strong feeding preference for humans as did sandflies for armadillos, thus presenting potential utility to further study related to host-vector interactions.

Biodiversity inventory remains limited in marine systems due to unbalanced access to the three ocean dimensions. The use of environmental DNA (eDNA) for metabarcoding allows fast and effective biodiversity inventory and is forecast as a future biodiversity research and biomonitoring tool. However, in poorly understood ecosystems, eDNA results remain difficult to interpret due to large gaps in reference databases and PCR bias limiting the detection of some major phyla. Here, we aimed to circumvent these limitations by avoiding PCR and recollecting larger DNA fragments to improve assignment of detected taxa through phylogenetic reconstruction. We applied capture by hybridization (CBH) to enrich DNA from deep-sea sediment samples and compared the results with those obtained through an up-to-date metabarcoding PCR-based approach (MTB). Originally developed for bacterial communities by targeting 16S rDNA, the CBH approach was applied to 18S rDNA to improve the detection of species forming benthic communities of eukaryotes, with particular focus on metazoans. The results confirmed the possibility of extending CBH to metazoans with two major advantages: i) CBH revealed a broader spectrum of prokaryotic, eukaryotic, and particularly metazoan diversity, and ii) CBH allowed much more robust phylogenetic reconstructions of full-length barcodes with up to 1900 base pairs. This is particularly important for taxa whose assignment is hampered by gaps in reference databases. This study provides a database and probes to apply 18S CBH to diverse marine systems, confirming this promising new tool to improve biodiversity assessments in data-poor ecosystems like those in the deep sea.

Pollinators are in decline thanks to the combined stresses of disease, pesticides, habitat loss, and climate. Honey bees face numerous pests and pathogens but arguably none are as devastating as Deformed wing virus (DWV). Understanding host-pathogen interactions and virulence of DWV in honey bees is slowed by the lack of cost-effective high-throughput screening methods for viral infection. Currently, analysis of virus infection in bees and their colonies is tedious, requiring a well-equipped molecular biology laboratory and the use of hazardous chemicals. Here we describe cDNA clones of DWV tagged with green fluorescent protein (GFP) or nanoluciferase (nLuc), providing high-throughput detection and quantification of virus infections. GFP fluorescence is recorded non-invasively in living bees via commonly available long-wave UV light sources and a smartphone camera or a standard ultraviolet transilluminator gel imaging system. Nonlethal monitoring with GFP allows high-throughput screening and serves as a direct breeding tool for identifying honey bee parents with increased antivirus resistance. Expression using the nLuc reporter strongly correlates with virus infection levels and is especially sensitive. Using multiple reporters, it is also possible to visualize competition, differential virulence, and host tissue targeting by co-occuring pathogens. Finally, it is possible to directly assess the risk of cross-species ‘spillover’ from honey bees to other pollinators and vice versa.

Metabarcoding is an important tool for understanding fungal communities. The internal transcribed spacer (ITS) rDNA is the accepted fungal barcode but has known problems. The large subunit (LSU) rDNA has also been used to investigate fungal communities but available LSU metabarcoding primers were mostly designed to target Dikarya (Ascomycota + Basidiomycota) with little attention to early diverging fungi (EDF). However, evidence from multiple studies suggests that EDF comprise a large portion of unknown diversity in community sampling. Here we investigate how DNA marker choice and methodological biases impact recovery of EDF from environmental samples. We focused on one EDF lineage, Zoopagomycota, as an example. We evaluated three primer sets (ITS1F/ITS2, LROR/LR3, and LR3 paired with new primer LR22F) to amplify and sequence a Zoopagomycota mock community and a set of 146 environmental samples with Illumina MiSeq. We compared two taxonomy assignment methods and created an LSU reference database compatible with AMPtk software. The two taxonomy assignment methods recovered strikingly different communities of fungi and EDF. Target fragment length variation exacerbated PCR amplification biases and influenced downstream taxonomic assignments, but this effect was greater for EDF than Dikarya. To improve identification of LSU amplicons we performed phylogenetic reconstruction and illustrate the advantages of this critical tool for investigating identified and unidentified sequences. Our results suggest much of the EDF community may be missed or misidentified with “standard” metabarcoding approaches and modified techniques are needed to understand the role of these taxa in a broader ecological context.

Phylogenetic trees have been extensively used in community ecology. However, how the phylogenetic reconstruction affects ecological inferences is poorly understood. In this study, we reconstructed three different types of phylogenetic trees (a synthetic-tree generated using VPhylomaker, a barcode-tree generated using rbcL+matK+trnH-psbA and a genome-tree generated from plastid genomes) that represented an increasing level of phylogenetic resolution among 580 woody plant species from six dynamic plots in subtropical evergreen broadleaved forests of China. We then evaluated the performance of each phylogeny in estimations of community phylogenetic structure, turnover and phylogenetic signal in functional traits. As expected, the genome-tree was most resolved and most supported for relationships among species. For local phylogenetic structure, the three trees showed consistent results with Faith’s PD and MPD; however, only the synthetic-tree produced significant clustering patterns using MNTD for some plots. For phylogenetic turnover, contrasting results between the molecular trees and the synthetic-tree occurred only with nearest neighbor distance. The barcode-tree agreed more with the genome-tree than the synthetic-tree for both phylogenetic structure and turnover. For functional traits, both the barcode-tree and genome-tree detected phylogenetic signal in maximum height, but only the genome-tree detected signal in leaf width. This is the first study that uses plastid genomes in large-scale community phylogenetics. Our results highlight the outperformance of genome-trees over barcode-trees and synthetic-trees for the analyses studied here. Our results also point to the possibility of Type I and II errors in estimation of phylogenetic structure and turnover and detection of phylogenetic signal when using synthetic-trees.

A high-quality reference genome is necessary to determine the molecular mechanisms underlying important biological phenomena; therefore, in the present study, a chromosome-level genome assembly of the Chinese shrimp Fenneropenaeus chinensis was performed. Muscle of a male shrimp was sequenced using PacBio platform, and assembled by Hi-C technology. The assembled F. chinensis genome was 1,465.32 Mb with contig N50 of 472.84 Kb, including 57.73% repetitive sequences, and was anchored to 43 pseudochromosomes, with scaffold N50 of 36.87 Mb. In total, 25,026 protein-coding genes were predicted. The genome size of F. chinensis showed significant contraction in comparison with that of other penaeid species, which is likely related to migration observed in this species. However, the F. chinensis genome included several expanded gene families related to cellular processes and metabolic processes, and the contracted gene families were associated with virus infection process. The findings signify the adaptation of F. chinensis to the selection pressure of migration and cold environment. Furthermore, the selection signature analysis identified genes associated with metabolism, phototransduction, and nervous system in cultured shrimps when compared with wild population, indicating targeted, artificial selection of growth, vision, and behavior during domestication. The construction of the genome of F. chinensis provided valuable information for the further genetic mechanism analysis of important biological processes, and will facilitate the research of genetic changes during evolution.

Here we present an annotated, chromosome-anchored, genome assembly for Lake Trout (Salvelinus namaycush) – a highly diverse salmonid species of notable conservation concern and an excellent model for research on adaptation and speciation. We leveraged Pacific Biosciences long-read sequencing, paired-end Illumina sequencing, proximity ligation (Hi-C), and a previously published linkage map to produce a highly contiguous assembly composed of 7,378 contigs (contig N50 = 1.8 mb) assigned to 4,120 scaffolds (scaffold N50 = 44.975 mb). 84.7% of the genome was assigned to 42 chromosome-sized scaffolds and 93.2% of Benchmarking Universal Single Copy Orthologs were recovered, putting this assembly on par with the best currently available salmonid genomes. Estimates of genome size based on k-mer frequency analysis were highly similar to the total size of the finished genome, suggesting that the entirety of the genome was recovered. A mitome assembly was also produced. Self-vs-self synteny analysis allowed us to identify homeologs resulting from the Salmonid specific autotetraploid event (Ss4R) and alignment with three other salmonid species allowed us to identify homologous chromosomes in other species. We also generated multiple resources useful for future genomic research on Lake Trout including a repeat library and a sex averaged recombination map. A novel RNA sequencing dataset was also used to produce a publicly available set of gene annotations using the National Center for Biotechnology Information Eukaryotic Genome Annotation Pipeline. Potential applications of these resources to population genetics and the conservation of native populations are discussed.

The promotion of responsible and sustainable trade in biological resources is widely proposed as one solution to mitigate currently high levels of global biodiversity loss. Various molecular identification methods have been proposed as appropriate tools for monitoring global supply chains of commercialized animals and plants. We demonstrate the efficacy of target capture genomic barcoding in identifying and establishing the geographic origin of samples traded as Anacyclus pyrethrum, a medicinal plant assessed as globally vulnerable in the IUCN Red List. Samples collected from national and international supply chains were identified through target capture sequencing of 443 low-copy nuclear makers and compared to results derived from genome skimming of plastome, standard plastid barcoding regions and ITS. Both target capture and genome skimming provided approximately 3.4 million reads per sample, but target capture largely outperformed standard plant DNA barcodes and entire plastid genome sequences. Despite the difficulty of distinguishing among closely related species and infraspecific taxa of Anacyclus using conventional taxonomic methods, we succeeded in identifying 89 of 110 analysed samples to subspecies level without ambiguity through target capture. Furthermore, we were able to discern the geographical origin of Anacyclus samples collected in Moroccan, Indian and Sri Lankan markets, differentiating between plant materials originally harvested from diverse populations in Algeria and Morocco. With a recent drop in the cost of analysing samples, target capture offers the potential to routinely identify commercialized plant species and determine their geographic origin. It promises to play an important role in monitoring and regulation of plant species in trade, supporting biodiversity conservation efforts, and in ensuring that plant products are unadulterated, contributing to consumer protection.

Until recently many historical museum specimens were largely inaccessible to genomic inquiry, but high-throughput sequencing (HTS) approaches have allowed researchers to successfully sequence genomic DNA from dried and fluid-preserved museum specimens. In addition to preserved specimens, many museums contain large series of allozyme supernatant samples but the amenability of these samples to HTS has not yet been assessed. Here, we compared the performance of a target-capture approach using alternative sources of genomic DNA from ten specimens of spring salamanders (Plethodontidae: Gyrinophilus porphyriticus) collected 1985–1990: allozyme supernatants, allozyme homogenate pellets, and formalin-fixed tissues. We designed capture probes based on double-digest restriction-site associated (RADseq) sequencing derived loci from seven of the specimens and assessed the success and consistency of capture and RADseq technical replicates. This study design enabled direct comparisons of data quality and potential biases among the different datasets for phylogenomic and population genomic analyses. We found that in phylogenetic analyses, all replicates for a given specimen clustered together, but in principal component space, RADseq replicates did not cluster with corresponding capture-based replicates. SNP calls were on average 18.3% different between technical replicates, but these discrepancies were primarily due to differences in heterozygous/homozygous SNP calls. We demonstrate that both allozyme supernatant and formalin-fixed samples can be successfully used for population genomic analyses and we discuss ways to identify and reduce biases associated with combining capture and RADseq data.

The soybean cyst nematode (Heterodera glycines) is a sedentary plant parasite that exceeds a billion dollars in yield losses annually. It has spread across the soybean-producing world, emerging as the primary pathogen of soybeans. This problem is exacerbated by H. glycines populations overcoming the limited sources of natural resistance in soybean and by the lack of effective and safe alternative treatments. Although there are genetic determinants that render soybean plants resistant to certain nematode genotypes, resistant soybean cultivars are increasingly ineffective because their multi-year usage has selected for virulent H. glycines populations. Successful H. glycines infection relies on the comprehensive re-engineering of soybean root cells into a syncytium, as well as the long-term suppression of host defenses to ensure syncytial viability. At the forefront of these complex molecular interactions are effectors, the proteins secreted by H. glycines into host root tissues. The mechanisms that control genomic effector acquisition, diversification, and selection are important insights needed for the development of essential novel control strategies. As a foundation to obtain this understanding, we developed a nine scaffold, 158Mb pseudomolecule assembly of the H. glycines genome using PacBio, Chicago, and Hi-C sequencing. An annotation of 22,465 genes was predicted using a Mikado pipeline informed by published short- and long-read expression data. Here we present results from our assembly and annotation of the H. glycines genome.

[Definitive version of this article may be found here] The mitochondrial gene cytochrome-c-oxidase subunit 1 (COI) is useful in many taxa for phylogenetics, population genetics, metabarcoding, and rapid species identifications. However, the phylum Ctenophora (comb jellies) has historically been difficult to study due to divergent mitochondrial sequences and the corresponding inability to amplify COI with degenerate and standard COI ‘barcoding’ primers. As a result, there are very few COI sequences available for ctenophores, despite over 200 described species in the phylum. Here, we designed new primers and amplified the COI fragment from members of all major groups of ctenophores, including many undescribed species. Phylogenetic analyses of the resulting COI sequences revealed high diversity within many groups that was not evident from more conserved 18S rDNA sequences, in particular among the Lobata. The COI phylogenetic results also revealed unexpected community structure within the genus Bolinopsis, suggested new species within the genus Bathocyroe, and supported the ecological and morphological differences of some species such as Lampocteis cruentiventer and similar lobates (Lampocteis sp. ‘V’ stratified by depth, and ‘A’ differentiated by color). The newly described primers reported herein provide important tools to enable researchers to illuminate the diversity of ctenophores worldwide via quick molecular identifications, improve the ability to analyze environmental DNA by improving reference libraries and amplifications, and enable a new breadth of population genetic studies.

Many model organisms have obtained a prominent status due to an advantageous combination of their life-history characteristics, genetic properties and also practical considerations. In non-crop plants, Arabidopsis thaliana is the most renowned model and has been used as study system to elucidate numerous biological processes at the molecular level. Once a complete genome sequence was available, research has markedly accelerated and further established A. thaliana as the reference to stimulate studies in other species with different biology. Within the Brassicaceae family, the arctic-alpine perennial Arabis alpina has become a model complementary to A. thaliana to study life-history evolution and ecological genomics in harsh environments. In this review, we provide an overview of the properties that facilitated the rapid emergence of A. alpina as a plant model. We summarize the evolutionary history of A. alpina, including the diversification of its mating system, and discuss recent progress in the molecular dissection of developmental traits that are related to its perennial life history and environmental adaptation. We indicate open questions from which future research might be developed in other Brassicaceae species or more distantly related plant families.

Identifying local adaptation in bottlenecked species is essential for conservation management. Selection detection methods have an important role in species management plans, assessments of adaptive capacity, and looking for responses to climate change. Yet, the allele frequency changes exploited in selection detection methods are similar to those caused by the strong neutral genetic drift expected during a bottleneck. Consequently, it is often unclear what accuracy selection detection methods have across bottlenecked populations. In this study, simulations were used to explore if signals of selection could be confidently distinguished from genetic drift across 23 bottlenecked and reintroduced populations of Alpine ibex (Capra ibex). The meticulously recorded demographic history of the Alpine ibex was used to generate comprehensive simulated SNP data. The simulated SNPs were then used to benchmark the confidence we could place in outliers identified in empirical Alpine ibex SNP data. Within the simulated dataset, the false positive rates were high for all selection detection methods but fell substantially when two or more methods were combined. True positive rates were consistently low and became negligible with increased stringency. Despite finding many outlier loci in the empirical Alpine ibex SNPs, none could be distinguished from genetic drift-driven false positives. Unfortunately, the low true positive rate also prevents the exclusion of recent local adaptation within the Alpine ibex. The baselines and stringent approach outlined here should be applied to other bottlenecked species to ensure the risk of false positive, or negative, signals of selection are accounted for in conservation management plans.

Mapping the genes underlying ecologically-relevant traits in natural populations is fundamental to develop a molecular understanding of species adaptation. Current sequencing technologies enable the characterisation of a species' genetic diversity across the landscape or even over its whole range. The relevant capture of the genetic diversity across the landscape is critical for a successful genetic mapping of traits and there are no clear guidelines on how to achieve an optimal sampling and which sequencing strategy to implement. Here we determine through simulation, the sampling scheme that maximises the power to map the genetic basis of a complex trait in an outbreeding species across an idealised landscape and draw genomic predictions for the trait, comparing individual and pool sequencing strategies. Our results show that QTL detection power and prediction accuracy are higher when more populations over the landscape are sampled and this is more cost-effectively done with pool sequencing than with individual sequencing. Additionally, we recommend sampling populations from areas of high genetic diversity. As progress in sequencing enables the integration of trait-based functional ecology into landscape genomics studies, these findings will guide study designs allowing direct measures of genetic effects in natural populations across the environment.