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Knowledge of global patterns of genetic diversity is essential for biodiversity conservation as this parameter describes the ability of a species to respond to environmental changes. Ichneumonoids parasitoid wasps are among the few taxa showing an anomalous latitudinal diversity gradient. Using the largest georeferenced molecular dataset for this group, we used a macrogenetics approach to examine latitudinal patterns and predictors of intraspecific genetic diversity. We calculated the mean nucleotide diversity of mitochondrial DNA barcode sequences at three geographic levels: grid cells, latitudinal bands and climatic zones. Nucleotide diversity values were consistently higher at northern temperate latitudes, peaking at 50°. We found a positive but weak relationship between intraspecific diversity and the latitude, between intra‐ and interspecific diversity, and a positive effect of the temperature range. Examining the spatial relationship between different levels of biodiversity and its drivers is particularly relevant considering climate change and its impact on species distribution. Yet, in insects, it has been challenging to integrate ecological, evolutionary and geographical components when analysing the processes leading to species richness gradients. [ABSTRACT FROM AUTHOR]

This open-access special issue features 12 full articles representing emerging trends from the international DNA barcoding community. Several articles highlight how DNA-based techniques are elucidating the species diversity, biogeography, and [...]

Introduction: Microbes are increasingly (re)considered for environmental assessments because they are powerful indicators for the health of ecosystems. The complexity of microbial communities necessitates powerful novel tools to derive conclusions for environmental decision-makers, and machine learning is a promising option in that context. While amplicon sequencing is typically applied to assess microbial communities, metagenomics and total RNA sequencing (herein summarized as omics-based methods) can provide a more holistic picture of microbial biodiversity at sufficient sequencing depths. Despite this advantage, amplicon sequencing and omics-based methods have not yet been compared for taxonomy-based environmental assessments with machine learning. Methods: In this study, we applied 16S and ITS-2 sequencing, metagenomics, and total RNA sequencing to samples from a stream mesocosm experiment that investigated the impacts of two aquatic stressors, insecticide and increased fine sediment deposition, on stream biodiversity. We processed the data using similarity clustering and denoising (only applicable to amplicon sequencing) as well as multiple taxonomic levels, data types, feature selection, and machine learning algorithms and evaluated the stressor prediction performance of each generated model for a total of 1,536 evaluated combinations of taxonomic datasets and data-processing methods. Results: Sequencing and data-processing methods had a substantial impact on stressor prediction. While omics-based methods detected a higher diversity of taxa than amplicon sequencing, 16S sequencing outperformed all other sequencing methods in terms of stressor prediction based on the Matthews Correlation Coefficient. However, even the highest observed performance for 16S sequencing was still only moderate. Omics-based methods performed poorly overall, but this was likely due to insufficient sequencing depth. Data types had no impact on performance while feature selection significantly improved performance for omicsbased methods but not for amplicon sequencing. Discussion: We conclude that amplicon sequencing might be a better candidate for machine-learning-based environmental stressor prediction than omics-based methods, but the latter require further research at higher sequencing depths to confirm this conclusion. More sampling could improve stressor prediction performance, and while this was not possible in the context of our study, thousands of sampling sites are monitored for routine environmental assessments, providing an ideal framework to further refine the approach for possible implementation in environmental diagnostics. [ABSTRACT FROM AUTHOR]

The small subunit (SSU) ribosomal RNA (rRNA) is the most commonly used marker for the identification of microbial taxa, but its full‐length reconstruction from high‐throughput sequencing (HTS) data remains challenging. Metagenomics and total RNA sequencing (total RNA‐Seq) are target‐PCR‐free HTS methods that are used to characterize microbial communities and simultaneously reconstruct SSU rRNA sequences. However, more testing is required to determine and improve their effectiveness.We processed metagenomics and total RNA‐Seq data retrieved from a commercially available mock microbial community and an aquarium sample using 112 combinations of data processing tools. We determined the SSU rRNA reconstruction completeness of both sequencing methods for both samples and analysed the impact of data processing tools on SSU rRNA completeness.In contrast to metagenomics, total RNA‐Seq allowed for the complete or near‐complete reconstruction of all mock community SSU rRNA sequences and generated up to 438 SSU rRNA sequences with ≥80% completeness from the aquarium sample using only 1/5 of an Illumina MiSeq run. SSU rRNA completeness of metagenomics significantly correlated with the genome size of mock community species. Data processing tools impacted SSU rRNA completeness, in particular the utilized assemblers.These results are promising for the high‐throughput reconstruction of novel full‐length SSU rRNA sequences and could advance the simultaneous application of multiple ‐omics approaches in routine environmental assessments to allow for more holistic assessments of ecosystems. [ABSTRACT FROM AUTHOR]

Abstract: The early life-history stages of fishes are poorly known, impeding acquisition of the identifications needed to monitor larval recruitment and year-class strength. A comprehensive database of COI sequences, linked [...]

The two special issues--Barcodes to Biomes--mark the one-year anniversary of the 6th International Barcode of Life Conference held in Guelph, Canada. The 6th Conference brought together 601 delegates from 51 [...]

DNA barcoding--the sequencing of short, standardized DNA regions for specimen identification and species discovery--has promised to facilitate rapid access to biodiversity knowledge by diverse users. Here, we advance our opinion that increased global participation in genetics research is beneficial, both to scientists and for science, and explore the premise that DNA barcoding can help to democratize participation in genetics research. We examine publication patterns (2003-2014) in the DNA barcoding literature and compare trends with those in the broader, related domain of genomics. While genomics is the older and much larger field, the number of nations contributing to the published literature is similar between disciplines. Meanwhile, DNA barcoding exhibits a higher pace of growth in the number of publications as well as greater evenness among nations in their proportional contribution to total authorships. This exploration revealed DNA barcoding to be a highly international discipline, with growing participation by researchers in especially biodiverse nations. We briefly consider several of the challenges that may hinder further participation in genetics research, including access to training and molecular facilities as well as policy relating to the movement of genetic resources. Keywords: DNA barcoding, International Barcode of Life, genomics, research trends, collaboration. Le codage a barres de l'ADN, soit le sequencage de courtes regions d'ADN standardisees pour des fins d'identification de specimens et la decouverte d'especes, promettait de faciliter un acces rapide aux connaissances sur la biodiversite par divers utilisateurs. Dans cet article, les auteurs mettent de l'avant qu'une participation internationale accrue a la recherche en genetique est benefique tant pour les chercheurs que pour la science et ils explorent l'hypothese que le codage a barres de l'ADN peut aider a democratiser la participation a la recherche en genetique. Les auteurs ont examine l'evolution des publications (2003-2014) dans le domaine du codage a barres de l'ADN et ont compare les tendances avec celles observees dans le domaine plus large mais apparente de la recherche en genomique. Bien que la genomique soit un champ de recherche plus ancien et beaucoup plus vaste, le nombre de pays ayant contribue a la litterature est semblable entre ces deux champs. De plus, le codage a barres de l'ADN presente une croissance plus rapide du nombre de publications ainsi qu'une plus grande uniformite entre pays en ce qui a trait a la contribution proportionnelle des auteurs. Cette exploration a revele que le codage a barres de l'ADN est une discipline hautement internationale, avec une participation croissante des chercheurs de pays presentant une biodiversite particulierement grande. Les auteurs discutent brievement de plusieurs des defis qui pourraient restreindre une participation accrue a la recherche en genetique, incluant l'acces a la formation et aux laboratoires d'analyse de meme que les politiques regissant le mouvement des ressources genetiques. [Traduit par la Redaction] Mots-cles: codage a barres de l'ADN, projet international du code barre du vivant, genomique, tendances en recherche, collaboration., The benefits of increasing global participation in genetics research The invitation to contribute an opinion piece to the special issue entitled 'Genomics: the Power and the Promise 2014' inspired us [...]

Although DNA barcodes-based operational taxonomic units (OTUs) are increasingly used in earthworm research, the relative efficiency of the different methods available to delimit them has not yet been tested on a comprehensive dataset. For this study, we used three datasets containing 651, 2304 and 4773 COI barcodes of earthworms from French Guiana, respectively, to compare five of these methods: two phylogenetic methods—namely Poisson Tree Processes (PTP) and General Mixed Yule Coalescence (GMYC)—and three distance matrix methods—namely Refined Single Linkage (RESL, used for assigning Barcode Index Numbers in the Barcode of Life Data systems), Automatic Barcode Gap Discovery (ABGD), and Assemble Species by Automatic Partitioning (ASAP). We found that phylogenetic approaches are less suitable for delineating OTUs from DNA barcodes in earthworms, especially for large sets of sequences. The computation times are unreasonable, they often fail to converge, and they also show a strong tendency to oversplit species. Among distance-based methods, RESL also has a clear tendency to oversplitting, while ABGD and ASAP are less prone to mismatches and have short computation times. ASAP requires less a priori knowledge for model parameterisation than AGBD, provides efficient graphical outputs, and has a much lower tendency to generate mismatches. [ABSTRACT FROM AUTHOR]

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The effective use of metabarcoding in biodiversity science has brought important analytical challenges due to the need to generate accurate taxonomic assignments. The assignment of sequences to genus or species level is critical for biodiversity surveys and biomonitoring, but it is particularly challenging as researchers must select the approach that best recovers information on species composition. This study evaluates the performance and accuracy of seven methods in recovering the species composition of mock communities by using COI barcode fragments. The mock communities varied in species number and specimen abundance, while upstream molecular and bioinformatic variables were held constant, and using a set of COI fragments. We evaluated the impact of parameter optimization on the quality of the predictions. Our results indicate that BLAST top hit competes well with more complex approaches if optimized for the mock community under study. For example, the two machine learning methods that were benchmarked proved more sensitive to reference database heterogeneity and completeness than methods based on sequence similarity. The accuracy of assignments was impacted by both species and specimen counts (query compositional heterogeneity) which ultimately influence the selection of appropriate software. We urge researchers to: (i) use realistic mock communities to allow optimization of parameters, regardless of the taxonomic assignment method employed; (ii) carefully choose and curate the reference databases including completeness; and (iii) use QIIME, BLAST or LCA methods, in conjunction with parameter tuning to better assign taxonomy to diverse communities, especially when information on species diversity is lacking for the area under study. see also the Perspective by Holly M. Bik. [ABSTRACT FROM AUTHOR]

Arthropod communities in buildings have not been extensively studied, although humans have always shared their homes with them. In this study we explored if arthropod DNA can be retrieved and metabarcoded from indoor environments through the collection of dead specimens in light fixtures to better understand what shapes arthropod diversity in our homes. Insects were collected from 45 light fixtures at the Centre for Biodiversity Genomics (CBG, Guelph, Canada), and by community scientists at 12 different residential homes in Southern Ontario. The CBG ground floor of the CBG showed the greatest arthropod diversity, especially in light fixtures that were continuously illuminated. The community scientist samples varied strongly by light fixture type, lightbulb used, time passed since lamp was last cleaned, and specimen size. In all cases, the majority of OTUs was not shared between samples even within the same building. This study demonstrates that light fixtures might be a useful resource to determine arthropod diversity in our homes, but individual samples are likely not representative of the full diversity. [ABSTRACT FROM AUTHOR]

Metabarcoding has become a common approach to the rapid identification of the species composition in a mixed sample. The majority of studies use established short‐read high‐throughput sequencing platforms. The Oxford Nanopore MinIONTM, a portable sequencing platform, represents a low‐cost alternative allowing researchers to generate sequence data in the field. However, a major drawback is the high raw read error rate that can range from 10% to 22%.To test whether the MinIONTM represents a viable alternative to other sequencing platforms, we used rolling circle amplification (RCA) to generate full‐length consensus DNA barcodes for a bulk mock sample of 50 aquatic invertebrate species with at least 15% genetic distance to each other. By applying two different laboratory protocols, we generated two MinIONTM runs that were used to build error‐corrected consensus sequences. A newly developed Python pipeline, ASHURE, was used for data processing, consensus building, clustering and taxonomic assignment of the resulting reads.Our pipeline achieved median accuracies of up to 99.3% for long concatemeric reads (>45 barcodes) and successfully identified all 50 species in the mock community. The use of RCA was integral for increasing consensus accuracy but was also the most time‐consuming step of the laboratory workflow. Most concatemeric reads were skewed towards a shorter read length range with a median read length of up to 1,262 bp.Our study demonstrates that Nanopore sequencing can be used for metabarcoding, but exploration of other isothermal amplification procedures to improve consensus accuracy is recommended. [ABSTRACT FROM AUTHOR]

Riparian forests are commonly advocated as a key management option to mitigate the effects of agriculture on headwater stream biodiversity and ecosystem functions. However, the benefits of riparian forests might be reduced by uninterrupted catchment-scale pollution. We studied the effects of riparian land use on multiple ecological endpoints in headwater streams in an agricultural landscape. We studied stream habitat characteristics, water temperature and algal accrual, and macrophyte, benthic macroinvertebrate and fish communities in 11 paired forested and open agricultural headwater stream reaches that differed in their extent of riparian forest cover but had similar water quality. Hydromorphological habitat quality was higher in forested reaches than in open reaches. Riparian forest had a strong effect on the summer water temperature regime, with maximum and mean water temperatures and temperature variation in forested reaches substantially lower than in open reaches. Macrophyte communities differed between forested and open reaches. The mean abundance of bryophytes was higher in forested reaches but the difference to open reaches was only marginally significant, whereas graminoids were significantly more abundant in open reaches. Within-stream dissimilarity of benthic macroinvertebrate community structure was significantly related to the difference in riparian land use between reach pairs. The relative DNA sequence abundance of pollution-sensitive Ephemeroptera, Plecoptera, and Trichoptera species tended to be higher in forested reaches than in open reaches. Finally, fish densities were not significantly different between forested and open reaches, although densities were higher in forested reaches. This unequivocal evidence for the ecological benefits of forested riparian reaches in agricultural headwater streams suggests that riparian forest can partly mitigate the adverse impacts of agricultural diffuse pollution on biota. The strong effect of forests on stream water temperature suggest that riparian forest could also mitigate harmful effects on headwater stream biodiversity and ecosystem functions of the predicted more frequent high summer temperatures. [ABSTRACT FROM AUTHOR]

Abstract Background The cichlid fishes in general, and the exceptionally diverse East African haplochromine cichlids in particular, are famous examples of adaptive radiation and explosive speciation. Here we report the collection and annotation of more than 12,000 expressed sequence tags (ESTs) generated from three different cDNA libraries obtained from the East African haplochromine cichlid species Astatotilapia burtoni and Metriaclima zebra. Results We first annotated more than 12,000 newly generated cichlid ESTs using the Gene Ontology classification system. For evolutionary analyses, we combined these ESTs with all available sequence data for haplochromine cichlids, which resulted in a total of more than 45,000 ESTs. The ESTs represent a broad range of molecular functions and biological processes. We compared the haplochromine ESTs to sequence data from those available for other fish model systems such as pufferfish (Takifugu rubripes and Tetraodon nigroviridis), trout, and zebrafish. We characterized genes that show a faster or slower rate of base substitutions in haplochromine cichlids compared to other fish species, as this is indicative of a relaxed or reinforced selection regime. Four of these genes showed the signature of positive selection as revealed by calculating Ka/Ks ratios. Conclusion About 22% of the surveyed ESTs were found to have cichlid specific rate differences suggesting that these genes might play a role in lineage specific characteristics of cichlids. We also conclude that the four genes with a Ka/Ks ratio greater than one appear as good candidate genes for further work on the genetic basis of evolutionary success of haplochromine cichlid fishes.

Abstract Background Evolution of the deuterostome lineage was accompanied by an increase in systematic complexity especially with regard to highly specialized tissues and organs. Based on the observation of an increased number of paralogous genes in vertebrates compared with invertebrates, two entire genome duplications (2R) were proposed during the early evolution of vertebrates. Most glycolytic enzymes occur as several copies in vertebrate genomes, which are specifically expressed in certain tissues. Therefore, the glycolytic pathway is particularly suitable for testing theories of the involvement of gene/genome duplications in enzyme evolution. Results We assembled datasets from genomic databases of at least nine vertebrate species and at least three outgroups (one deuterostome and two protostomes), and used maximum likelihood and Bayesian methods to construct phylogenies of the 10 enzymes of the glycolytic pathway. Through this approach, we intended to gain insights into the vertebrate specific evolution of enzymes of the glycolytic pathway. Many of the obtained gene trees generally reflect the history of two rounds of duplication during vertebrate evolution, and were in agreement with the hypothesis of an additional duplication event within the lineage of teleost fish. The retention of paralogs differed greatly between genes, and no direct link to the multimeric structure of the active enzyme was found. Conclusion The glycolytic pathway has subsequently evolved by gene duplication and divergence of each constituent enzyme with taxon-specific individual gene losses or lineage-specific duplications. The tissue-specific expression might have led to an increased retention for some genes since paralogs can subdivide the ancestral expression domain or find new functions, which are not necessarily related to the original function.

Abstract Background Gene and genome duplication events increase the amount of genetic material that might then contribute to an increase in the genomic and phenotypic complexity of organisms during evolution. Thus, it has been argued that there is a relationship between gene copy number and morphological complexity and/or species diversity. This hypothesis implies that duplicated genes have subdivided or evolved novel functions compared to their pre-duplication proto-orthologs. Such a functional divergence might be caused by an increase in evolutionary rates in one ortholog, by changes in expression, regulatory evolution, insertion of repetitive elements, or due to positive Darwinian selection in one copy. We studied a set of 2466 genes that were present in Danio rerio, Takifugu rubripes, Tetraodon nigroviridis and Oryzias latipes to test (i) for forces of positive Darwinian selection; (ii) how frequently duplicated genes are retained, and (iii) whether novel gene functions might have evolved. Results 25% (610) of all investigated genes show significantly smaller or higher genetic distances in the genomes of particular fish species compared to their human ortholog than their orthologs in other fish according to relative rate tests. We identified 49 new paralogous pairs of duplicated genes in fish, in which one of the paralogs is under positive Darwinian selection and shows a significantly higher rate of molecular evolution in one of the four fish species, whereas the other copy apparently did not undergo adaptive changes since it retained the original rate of evolution. Among the genes under positive Darwinian selection, we found a surprisingly high number of ATP binding proteins and transcription factors. Conclusion The significant rate difference suggests that the function of these rate-changed genes might be essential for the respective fish species. We demonstrate that the measurement of positive selection is a powerful tool to identify divergence rates of duplicated genes and that this method has the capacity to identify potentially interesting candidates for adaptive gene evolution.

Abstract Background Costly structures need to represent an adaptive advantage in order to be maintained over evolutionary times. Contrary to many other conspicuous shell ornamentations of gastropods, the haired shells of several Stylommatophoran land snails still lack a convincing adaptive explanation. In the present study, we analysed the correlation between the presence/absence of hairs and habitat conditions in the genus Trochulus in a Bayesian framework of character evolution. Results Haired shells appeared to be the ancestral character state, a feature most probably lost three times independently. These losses were correlated with a shift from humid to dry habitats, indicating an adaptive function of hairs in moist environments. It had been previously hypothesised that these costly protein structures of the outer shell layer facilitate the locomotion in moist habitats. Our experiments, on the contrary, showed an increased adherence of haired shells to wet surfaces. Conclusion We propose the hypothesis that the possession of hairs facilitates the adherence of the snails to their herbaceous food plants during foraging when humidity levels are high. The absence of hairs in some Trochulus species could thus be explained as a loss of the potential adaptive function linked to habitat shifts.

High-throughput sequencing (HTS) technologies have opened new avenues for the study of biodiversity. This innovation has resulted in an exponentially increasing number of concepts, methods, and research studies in the [...]

DNA barcoding opens new perspectives on the way we document biodiversity. Initially proposed to circumvent the limits of morphological characters to assign unknown individuals to known species, DNA barcoding has been used in a wide array of studies where collecting species identity constitutes a crucial step. The assignment of unknowns to knowns assumes that species are already well identified and delineated, making the assignment performed reliable. Here, we used DNA‐based species delimitation and specimen assignment methods iteratively to tackle the inventory of the Indo‐Australian Archipelago grey mullets, a notorious case of taxonomic complexity that requires DNA‐based identification methods considering that traditional morphological identifications are usually not repeatable and sequence mislabeling is common in international sequence repositories. We first revisited a DNA barcode reference library available at the global scale for Mugilidae through different DNA‐based species delimitation methods to produce a robust consensus scheme of species delineation. We then used this curated library to assign unknown specimens collected throughout the Indo‐Australian Archipelago to known species. A second iteration of OTU delimitation and specimen assignment was then performed. We show the benefits of using species delimitation and specimen assignment methods iteratively to improve the accuracy of specimen identification and propose a workflow to do so. [ABSTRACT FROM AUTHOR]

The Coral Triangle (CT), a region spanning across Indonesia and Philippines, is home to about 4,350 marine fish species and is among the world's most emblematic regions in terms of conservation. Threatened by overfishing and oceans warming, the CT fisheries have faced drastic declines over the last decades. Usually monitored through a biomass‐based approach, fisheries trends have rarely been characterized at the species level due to the high number of taxa involved and the difficulty to accurately and routinely identify individuals to the species level. Biomass, however, is a poor proxy of species richness, and automated methods of species identification are required to move beyond biomass‐based approaches. Recent meta‐analyses have demonstrated that species richness peaks at intermediary levels of biomass. Consequently, preserving biomass is not equal to preserving biodiversity. We present the results of a survey to estimate the shore fish diversity retailed at the harbor of Ambon Island, an island located at the center of the CT that display exceptionally high biomass despite high levels of threat, while building a DNA barcode reference library of CT shore fishes targeted by artisanal fisheries. We sampled 1,187 specimens and successfully barcoded 696 of the 760 selected specimens that represent 202 species. Our results show that DNA barcodes were effective in capturing species boundaries for 96% of the species examined, which opens new perspectives for the routine monitoring of the CT fisheries. [ABSTRACT FROM AUTHOR]

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids biodiversity and ecosystem assessment. However, it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. This study tests the performance of 36 primer sets on a mock community containing 374 insect species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets were also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from both mock community and Malaise trap sample metabarcoding were used to select four primer sets for additional evaluation at different annealing temperatures (40–60 °C) using the mock community. The effect of temperature varied by primer pair but overall it only had a minor effect on taxon recovery. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrates that certain primer sets can recover most taxa in a diverse species assemblage. Thus, based our experimental set up, there is no need to employ several primer sets targeting the same gene region. We identify several suitable primer sets for arthropod metabarcoding, and specifically recommend BF3 + BR2, as it is not affected by primer slippage and provides maximal taxonomic resolution. The fwhF2 + fwhR2n primer set amplifies a shorter fragment and is therefore ideal when targeting degraded DNA (e.g., from gut contents). [ABSTRACT FROM AUTHOR]

Although DNA metabarcoding is an attractive approach for monitoring biodiversity, it is often difficult to detect all the species present in a bulk sample. In particular, sequence recovery for a given species depends on its biomass and mitome copy number as well as the primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 species. We used this community to examine how species recovery was impacted when amplicon pools were constructed in four ways. The first two protocols involved the construction of bulk DNA extracts from different body segments (Bulk Abdomen, Bulk Leg). The other protocols involved the production of DNA extracts from single legs which were then merged prior to PCR (Composite Leg) or PCR‐amplified separately (Single Leg) and then pooled. The amplicons generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5). The choice of sequencing platform did not substantially influence species recovery, although the Miseq delivered the highest sequence quality. As expected, species recovery was most efficient from the Single Leg treatment because amplicon abundance varied little among taxa. Among the three treatments where PCR occurred after pooling, the Bulk Abdomen treatment produced a more uniform read abundance than the Bulk Leg or Composite Leg treatment. Primer choice also influenced species recovery and evenness. Our results reveal how variation in protocols can have substantial impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote. [ABSTRACT FROM AUTHOR]

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the recent years. The method has matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high‐throughput sequencing capacity. However, workflows and sample tagging need to be optimised to accommodate for hundreds of samples within a single sequencing run.Here, we conceptualise a streamlined metabarcoding workflow, in which samples are processed in 96‐well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2 + BR2 primer pair up to three 96‐well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices, tagging can be extended to thousands of samples.We hope that our metabarcoding workflow will be used as a practical guide for future large‐scale biodiversity assessments involving freshwater invertebrates. However, as this is just one possible metabarcoding approach, we hope this article will stimulate discussion and publication of alternatives and extensions to this method. [ABSTRACT FROM AUTHOR]

African sponges, particularly freshwater sponges, are understudied relative to demosponges in most other geographical regions. Freshwater sponges (Spongillida) likely share a common ancestor; however, their evolutionary history, particularly during their radiation into endemic and allegedly cosmopolitan groups, is unclear. Freshwater sponges of at least 58 species of 17 genera and four families are described from Central and Eastern Africa, but the diversity is underestimated due to limited distinguishable morphological features. The discovery of additional cryptic species is very likely with the use of molecular techniques such as DNA barcoding. The Royal Museum of Central Africa (MRAC, Tervuren, Belgium) hosts one of the largest collections of (Central) African freshwater sponge type material. Type specimens in theory constitute ideal targets for molecular taxonomy; however, the success is frequently hampered by DNA degradation and deamination, which are a consequence of suboptimal preservation techniques. Therefore, we genotyped African demosponge holotype material of the MRAC with specific short primers suitable for degenerated tissue and compare the results with the current, morphology-based classification. Our results demonstrate the utility of minimalistic barcodes for identification of sponges, potentially enabling efficient identification of individuals in taxonomic or metabarcoding studies, and highlight inconsistencies in the current freshwater sponge classification. [ABSTRACT FROM AUTHOR]

Sustainably feeding the next generation is often described as one of the most pressing “grand challenges” facing the 21st century. Generally, scholars propose addressing this problem by increasing agricultural production, investing in technology to boost yields, changing diets, or reducing food waste. In this paper, we explore whether global food production is nutritionally balanced by comparing the diet that nutritionists recommend versus global agricultural production statistics. Results show that the global agricultural system currently overproduces grains, fats, and sugars while production of fruits and vegetables and protein is not sufficient to meet the nutritional needs of the current population. Correcting this imbalance could reduce the amount of arable land used by agriculture by 51 million ha globally but would increase total land used for agriculture by 407 million ha and increase greenhouse gas emissions. For a growing population, our calculations suggest that the only way to eat a nutritionally balanced diet, save land and reduce greenhouse gas emissions is to consume and produce more fruits and vegetables as well as transition to diets higher in plant-based protein. Such a move will help protect habitats and help meet the Sustainable Development Goals. [ABSTRACT FROM AUTHOR]

Background: DNA metabarcoding is used to generate species composition data for entire communities. However, sequencing errors in high-throughput sequencing instruments are fairly common, usually requiring reads to be clustered into operational taxonomic units (OTUs), losing information on intraspecific diversity in the process. While Cytochrome c oxidase subunit I (COI) haplotype information is limited in resolving intraspecific diversity it is nevertheless often useful e.g. in a phylogeographic context, helping to formulate hypotheses on taxon distribution and dispersal. Methods: This study combines sequence denoising strategies, normally applied in microbial research, with additional abundance-based filtering to extract haplotype information from freshwater macroinvertebrate metabarcoding datasets. This novel approach was added to the R package "JAMP" and can be applied to COI amplicon datasets. We tested our haplotyping method by sequencing (i) a single-species mock community composed of 31 individuals with 15 different haplotypes spanning three orders of magnitude in biomass and (ii) 18 monitoring samples each amplified with four different primer sets and two PCR replicates. Results: We detected all 15 haplotypes of the single specimens in the mock community with relaxed filtering and denoising settings. However, up to 480 additional unexpected haplotypes remained in both replicates. Rigorous filtering removes most unexpected haplotypes, but also can discard expected haplotypes mainly from the small specimens. In the monitoring samples, the different primer sets detected 177-200 OTUs, each containing an average of 2.40-3.30 haplotypes per OTU. The derived intraspecific diversity data showed population structures that were consistent between replicates and similar between primer pairs but resolution depended on the primer length. A closer look at abundant taxa in the dataset revealed various population genetic patterns, e.g. the stonefly Taeniopteryx nebulosa and the caddisfly Hydropsyche pellucidula showed a distinct north-south cline with respect to haplotype distribution, while the beetle Oulimnius tuberculatus and the isopod Asellus aquaticus displayed no clear population pattern but differed in genetic diversity. Discussion: We developed a strategy to infer intraspecific genetic diversity from bulk invertebrate metabarcoding data. It needs to be stressed that at this point this metabarcoding-informed haplotyping is not capable of capturing the full diversity present in such samples, due to variation in specimen size, primer bias and loss of sequence variants with low abundance. Nevertheless, for a high number of species intraspecific diversity was recovered, identifying potentially isolated populations and taxa for further more detailed phylogeographic investigation. While we are currently lacking large-scale metabarcoding datasets to fully take advantage of our new approach, metabarcoding-informed haplotyping holds great promise for biomonitoring efforts that not only seek information about species diversity but also underlying genetic diversity. [ABSTRACT FROM AUTHOR]