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Our ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation. Here, we used complementation of auxotrophs and DNA barcode sequencing (Coaux-Seq) to enable high-throughput characterization of protein function. Fragment libraries from eleven genetically diverse bacteria were tested in twenty different auxotrophic strains of Escherichia coli to identify genes that complement missing biochemical activity. We recovered 41% of expected hits, with effectiveness ranging per source genome, and observed success even with distant E. coli relatives like Bacillus subtilis and Bacteroides thetaiotaomicron. Coaux-Seq provided the first experimental validation for 53 proteins, of which 11 are less than 40% identical to an experimentally characterized protein. Among the unexpected function identified was a sulfate uptake transporter, an O-succinylhomoserine sulfhydrylase for methionine synthesis, and an aminotransferase. We also identified instances of cross-feeding wherein protein overexpression and nearby non-auxotrophic strains enabled growth. Altogether, Coaux-Seq's utility is demonstrated, with future applications in ecology, health, and engineering. Synopsis: Coaux-seq is a new method for high-throughput protein characterization, utilizing complementation of auxotrophs and next generation sequencing. The use of simple barcode sequencing (BarSeq) allowed for cost-effective and simple screening of 11 different genome fragment libraries across 20 different auxotrophic strains. The first experimental evidence of function is provided for 53 proteins. Unexpected functions were identified in three enzymes, including a sulfate uptake transporter from the TauE family, an O-succinylhomoserine sulfhydrylase for methionine synthesis, and an aminotransferase. Workflow optimizations and discussions on potential pitfalls are provided. Coaux-seq is a new method for high-throughput protein characterization, utilizing complementation of auxotrophs and next generation sequencing. [ABSTRACT FROM AUTHOR]

Ectomycorrhizal (ECM) fungi are important tree symbionts within forests. The biogeography of ECM fungi remains to be investigated because it is challenging to observe and identify species. Because most ECM plant taxa have a Holarctic distribution, it is difficult to evaluate the extent to which host preference restricts the global distribution of ECM fungi. To address this issue, we aimed to assess whether host preference enhances the endemism of ECM fungi that inhabit dipterocarp rainforests. Highly similar sequences of 175 operational taxonomic units (OTUs) for ECM fungi that were obtained from Lambir Hill's National Park, Sarawak, Malaysia, were searched for in a nucleotide sequence database. Using a two‐step binomial model, the probability of presence for the query OTUs and the registration rate of barcode sequences in each country were simultaneously estimated. The results revealed that the probability of presence in the respective countries increased with increasing species richness of Dipterocarpaceae and decreasing geographical distance from the study site (i.e. Lambir). Furthermore, most of the ECM fungi were shown to be endemic to Malaysia and neighbouring countries. These findings suggest that not only dispersal limitation but also host preference are responsible for the high endemism of ECM fungi in dipterocarp rainforests. Moreover, host preference likely determines the areas where ECM fungi potentially expand and dispersal limitation creates distance–decay patterns within suitable habitats. Although host preference has received less attention than dispersal limitation, our findings support that host preference has a profound influence on the global distribution of ECM fungi. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) metabarcoding has the potential to significantly improve surveys of biodiversity in freshwater systems. However, this methodology is still infrequently used in global hotspots of endemic species, in part because of two major barriers to the success of eDNA metabarcoding: (1) insufficient regional taxonomic representation in public reference sequence databases; and (2) inconsistent species incidence and abundance estimates when compared to conventional surveys.We sampled eDNA and conducted visual surveys in the headwaters of two rivers in the Cape Fold aquatic ecoregion, South Africa. A reference sequence database was generated for the regional diversity of fishes to improve taxonomic classification of endemic species. We also compared the consistency of incidence data and relative abundance estimates of fishes from eDNA metabarcoding sequencing results and visual surveys (snorkel and underwater cameras) at each site sampled.Only 1% of eDNA metabarcoding reads could be classified to fish species without the supplementation of reference sequence databases for local endemic species. Once regional reference sequences were added, a total of nine species were detected and >99% reads classified. A strong positive relationship (Φ = 0.75) was observed between the patterns of detections using eDNA and visual approaches. However, eDNA metabarcoding detected more species than visual methods. In addition, the relative read frequency and abundance observed in visual surveys was significantly correlated (R2 = 0.85–0.88, p < 0.001) in the majority of the small pools surveyed.Incomplete public reference sequence databases hinder the use of eDNA metabarcoding in regions of high endemicity. Local reference sequence libraries can overcome these challenges. When appropriately implemented, eDNA metabarcoding shows promise, producing results consistent or better than the more frequently used and labour‐intensive visual surveys.Efficient survey methods like eDNA metabarcoding are urgently needed to improve aquatic monitoring efforts globally. Understanding how eDNA metabarcoding sequencing results relate to conventional survey methods is a key step in its implementation in ecoregions with high endemicity. [ABSTRACT FROM AUTHOR]

Aegla is a genus of freshwater anomuran crabs encompassing 94 described species occurring in meridional South America. Brazil harbours the greatest diversity of species, mainly in its southern region, which becomes a priority area for Aegla conservation due to high species richness and phylogenetic diversity, elevated endemism and threats to the Atlantic Forest. Here, we assessed Aegla richness and distribution in this priority area and tested the hypothesis of crypticism in the region's species. Sampling sites were distributed inside and in the surroundings of five protected areas, covering the major distribution range of several species described for southern Brazil. We applied three species delimitation methods to COI mitochondrial gene data, coupled with phylogenetic and morphological analyses. By integrating species delimitation based on the COI data and traditional taxonomy, we uncovered a high proportion of hidden diversity among Aegla in our study area and identified ten known species besides impressive 17 new putative species. Of these, eight are possible new taxonomic units that do not conform to any known species, and nine are divided into three cryptic groups: Aegla jarai complex, Aegla franciscana complex and Aegla camargoi complex. Most of these species have a narrow spatial distribution, making them highly vulnerable to habitat degradation. Despite the real richness of the genus still being largely unknown, the high diversity revealed here makes our study area the richest known region regarding the number of Aegla species within its distribution, making this area even more worthy for conservation efforts. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

More efficient methods for extensive biodiversity monitoring are required to support rapid measures to address the biodiversity crisis. While environmental DNA (eDNA) metabarcoding and quantitative PCR (qPCR) methods offer advantages over traditional monitoring approaches, their large‐scale application is limited by the time and labour required for developing assays and/or for analysis. CRISPR (clustered regularly interspaced short palindromic repeats) diagnostic technologies (Dx) may overcome some of these limitations, but they have been used solely with species‐specific primers, restricting their versatility for biodiversity monitoring. Here, we demonstrate the feasibility of designing species‐specific CRISPR‐Dx assays in silico within a short metabarcoding fragment using a general primer set, a methodology we term 'ampliscanning', for 18 of the 22 amphibian species in Switzerland. We sub‐selected nine species, including three classified as regionally endangered, to test the methodology using eDNA sampled from ponds at nine sites. We compared the ampliscanning detections to data from traditional monitoring at these sites. Ampliscanning was successful at detecting target species with different prevalences across the landscape. With only one visit, we detected more species per site than three traditional monitoring visits (visual and acoustic detections by trained experts), in particular more elusive species and previously undocumented but expected populations. Ampliscanning detected 25 species/site combinations compared to 12 with traditional monitoring. Sensitivity analyses showed that larger numbers of field visits and PCR replicates are more important for reliable detection than many technical replicates at the CRISPR‐Dx assay level. Given the reduced sampling and analysis effort, our results highlight the benefits of eDNA and CRISPR‐Dx combined with universal primers for large‐scale monitoring of multiple endangered species across landscapes to inform conservation measures. [ABSTRACT FROM AUTHOR]

Efficient and accurate classification of DNA barcode data is crucial for large‐scale fungal biodiversity studies. However, existing methods are either computationally expensive or lack accuracy. Previous research has demonstrated the potential of deep learning in this domain, successfully training neural networks for biological sequence classification. We introduce the MycoAI Python package, featuring various deep learning models such as BERT and CNN tailored for fungal Internal Transcribed Spacer (ITS) sequences. We explore different neural architecture designs and encoding methods to identify optimal models. By employing a multi‐head output architecture and multi‐level hierarchical label smoothing, MycoAI effectively generalizes across the taxonomic hierarchy. Using over 5 million labelled sequences from the UNITE database, we develop two models: MycoAI‐BERT and MycoAI‐CNN. While we emphasize the necessity of verifying classification results by AI models due to insufficient reference data, MycoAI still exhibits substantial potential. When benchmarked against existing classifiers such as DNABarcoder and RDP on two independent test sets with labels present in the training dataset, MycoAI models demonstrate high accuracy at the genus and higher taxonomic levels, with MycoAI‐CNN being the fastest and most accurate. In terms of efficiency, MycoAI models can classify over 300,000 sequences within 5 min. We publicly release the MycoAI models, enabling mycologists to classify their ITS barcode data efficiently. Additionally, MycoAI serves as a platform for developing further deep learning‐based classification methods. The source code for MycoAI is available under the MIT Licence at https://github.com/MycoAI/MycoAI. [ABSTRACT FROM AUTHOR]

The democratisation of next‐generation sequencing has vastly increased the availability of sequencing data from metabarcoding. However, to effectively prepare these metabarcoding data for subsequent analysis, researchers must consistently apply several different bioinformatic tools—including those which denoise reads, cluster sequences and assign taxonomic identities. This often creates a bioinformatics bottleneck in workflows for non‐specialists due to obstacles around: (a) integrating different tools, (b) the inability to easily modify and rerun bioinformatic pipelines involving non‐scripted ('point‐and‐click') elements and (c) the multiple outputs that may be required of a single dataset (e.g. amplicon sequence variants [ASVs] and operational taxonomic units [OTUs]), which often results in users running pipelines multiple times.Here, we introduce SimpleMetaPipeline, an open‐source bioinformatics pipeline implemented in R, which addresses these obstacles. SimpleMetaPipeline integrates the most robust and commonly used existing bioinformatic tools in a single reproducible pipeline, with a streamlined choice of parameters, to generate a sequence data table containing alternative clustering and assignment options. SimpleMetaPipeline accepts demultiplexed paired‐end and single reads from multiple sequencing runs.We describe the pipeline and demonstrate how alternative annotations enable the easy implementation of multi‐algorithm agreement tests to strengthen inferences.SimpleMetaPipeline represents a valuable addition to the existing library of pipelines, providing easy and reproducible bioinformatics, including a range of commonly desired clustering and assignment options, such as OTUs and ASVs. [ABSTRACT FROM AUTHOR]

ABSTRACT Low coverage ‘genome‐skims’ are often used to assemble organelle genomes and ribosomal gene sequences for cost‐effective phylogenetic and barcoding studies. Natural history collections hold invaluable biological information, yet poor preservation resulting in degraded DNA often hinders polymerase chain reaction‐based analyses. However, it is possible to generate libraries and sequence the short fragments typical of degraded DNA to generate genome‐skims from museum collections. Here we introduce a snakemake toolkit comprised of three pipelines skim2mito, skim2rrna and gene2phylo, designed to unlock the genomic potential of historical museum specimens using genome skimming. Specifically, skim2mito and skim2rrna perform the batch assembly, annotation and phylogenetic analysis of mitochondrial genomes and nuclear ribosomal genes, respectively, from low‐coverage genome skims. The third pipeline gene2phylo takes a set of gene alignments and performs phylogenetic analysis of individual genes, partitioned analysis of concatenated alignments and a phylogenetic analysis based on gene trees. We benchmark our pipelines with simulated data, followed by testing with a novel genome skimming dataset from both recent and historical solariellid gastropod samples. We show that the toolkit can recover mitochondrial and ribosomal genes from poorly preserved museum specimens of the gastropod family Solariellidae, and the phylogenetic analysis is consistent with our current understanding of taxonomic relationships. The generation of bioinformatic pipelines that facilitate processing large quantities of sequence data from the vast repository of specimens held in natural history museum collections will greatly aid species discovery and exploration of biodiversity over time, ultimately aiding conservation efforts in the face of a changing planet. [ABSTRACT FROM AUTHOR]

The soybean pod borer, Leguminivora glycinivorella (Matsumura), is an important tortricid pest species widely distributed in most parts of China and its adjacent regions. Here, we analyzed the genetic diversity and population differentiation of L. glycinivorella using diverse genetic information including the standard cox1 barcode sequences, mitochondrial genomes (mitogenomes), and single‐nucleotide polymorphisms (SNPs) from genotyping‐by‐sequencing. Based on a comprehensive sampling (including adults or larvae of L. glycinivorella newly collected at 22 of the total 30 localities examined) that covers most of the known distribution range of this pest, analyses of 543 cox1 barcode sequences and 60 mitogenomes revealed that the traditionally recognized and widely distributed L. glycinivorella contains two sympatric and widely distributed genetic lineages (A and B) that were estimated to have diverged ∼1.14 million years ago during the middle Pleistocene. Moreover, low but statistically significant correlations were recognized between genetic differentiation and geographic or environmental distances, indicating the existence of local adaptation to some extent. Based on SNPs, phylogenetic inference, principal component analysis, fixation index, and admixture analysis all confirm the two divergent sympatric lineages. Compared with the stable demographic history of Lineage B, the expansion of Lineage A had possibly made the secondary contact of the two lineages probable, and this process may be driven by the climate fluctuation during the late Pleistocene as revealed by ecological niche modeling. [ABSTRACT FROM AUTHOR]

Holoplanktonic organisms are thought to have enormous populations sizes, expansive geographic ranges, and low species diversity. Previous work suggests that pelagic gastropods are no exception to this generalization. However, most of these data are derived from temperate species, whereas the bulk of pteropod diversity occurs in the tropics. Here, we present DNA barcode data for COI focused on the under‐studied limacinoid, gymnosome, and pseudothecosome pteropods collected from the coastal waters of Panama. We applied four molecular species delimitation approaches to determine the number of operational taxonomic units (OTUs) identifiable from our sequences and all data available from Barcode of Life Database (BOLD) and GenBank and compared these to the Barcode Index Number generated by the BOLD. Assemble Species by Automatic Partitioning (ASAP), Poisson Tree Processes (PTP), Bayesian Poisson Tree Processes (bPTP), and Generalized Mixed Yule Coalescent (GMYC) methods generally gave congruent results and suggest that pteropod diversity in the tropics is significantly underrepresented both in DNA sequence databases and in the number of named OTUs. Of 42 specimens collected and sequenced, we recovered 16 OTUs, only one of which belonged to an OTU already represented in sequence databases. Neighbor‐joining trees including the previously published sequences show that in all three groups morphospecies can include genetically divergent lineages. Many very divergent (>15% from nearest neighbor) taxa are also still represented by only a single sequence, suggesting that there is a large amount of cryptic or pseudo‐cryptic diversity still to be described. To aid this future endeavor, we include some preliminary 16S data derived from new pteropod‐specific primers. [ABSTRACT FROM AUTHOR]

Consumers are thought to select food resources based on their nutritional content. While laboratory experiments have explored this, the nutritional dynamics of invertebrate predators have been scarcely studied in the field given various methodological constraints. The intersection of these nutritional dynamics with predator traits is also poorly characterised, leading to many gaps in our understanding of how different predators forage and feed in natural systems. Here, we integrate dietary metabarcoding with prey macronutrient (protein, lipid and carbohydrate) content and abundance to assess how nutrients and predator traits (sex, life stage and taxonomy) interactively drive prey preferences in the field, using spider–prey interactions as a model system. Different spider genera, sexes and life stages had nutritionally distinct diets. Our analyses demonstrated disproportionate foraging (selection and avoidance) for prey rich in different macronutrients, with the nature of these relationships differing between spider taxa, life stages and sexes. This may be explained by niche differentiation among spider groups, driven by biases toward prey rich in different nutrients, or nutrient‐specific foraging in which individual spiders vary their nutritional preferences to redress deficits, although further evidence is required to confirm this. This insight into the nutritional dynamics of generalist invertebrate predators extends our understanding beyond lab‐based behavioural assays and provides a novel framework for other complex real‐world systems. [ABSTRACT FROM AUTHOR]

ABSTRACT Chironomidae, so‐called non‐biting midges, are considered key bioindicators of aquatic ecosystem variability. Data derived from morphologically identifying their chitinous remains in sediments document chironomid larvae assemblages, which are studied to reconstruct ecosystem changes over time. Recent developments in sedimentary DNA (sedDNA) research have demonstrated that molecular techniques are suitable for determining past and present occurrences of organisms. Nevertheless, sedDNA records documenting alterations in chironomid assemblages remain largely unexplored. To close this gap, we examined the applicability of sedDNA metabarcoding to identify Chironomidae assemblages in lake sediments by sampling and processing three 21–35 cm long sediment cores from Lake Sempach in Switzerland. With a focus on developing analytical approaches, we compared an invertebrate‐universal (FWH) and a newly designed Chironomidae‐specific metabarcoding primer set (CH) to assess their performance in detecting Chironomidae DNA. We isolated and identified chitinous larval remains and compared the morphotype assemblages with the data derived from sedDNA metabarcoding. Results showed a good overall agreement of the morphotype assemblage‐specific clustering among the chitinous remains and the metabarcoding datasets. Both methods indicated higher chironomid assemblage similarity between the two littoral cores in contrast to the deep lake core. Moreover, we observed a pronounced primer bias effect resulting in more Chironomidae detections with the CH primer combination compared to the FWH combination. Overall, we conclude that sedDNA metabarcoding can supplement traditional remain identifications and potentially provide independent reconstructions of past chironomid assemblage changes. Furthermore, it has the potential of more efficient workflows, better sample standardisation and species‐level resolution datasets. [ABSTRACT FROM AUTHOR]

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]

ABSTRACT It is a global priority to better manage the biosphere, but action must be informed by comprehensive data on the abundance and distribution of species. The acquisition of such information is currently constrained by high costs. DNA barcoding can speed the registration of unknown animal species, the most diverse kingdom of eukaryotes, as the BIN system automates their recognition. However, inexpensive sequencing protocols are critical as the census of all animal species is likely to require the analysis of a billion or more specimens. Barcoding involves DNA extraction followed by PCR and sequencing with the last step dominating costs until 2017. By enabling the sequencing of highly multiplexed samples, the Sequel platforms from Pacific BioSciences slashed costs by 90%, but these instruments are only deployed in core facilities because of their expense. Sequencers from Oxford Nanopore Technologies provide an escape from high capital and service costs, but their low sequence fidelity has, until recently, constrained adoption. However, the improved performance of its latest flow cells (R10.4.1) erases this barrier. This study demonstrates that a MinION flow cell can characterise an amplicon pool derived from 100,000 specimens while a Flongle flow cell can process one derived from several thousand. At $0.01 per specimen, DNA sequencing is now the least expensive step in the barcode workflow. [ABSTRACT FROM AUTHOR]

Astroblepus species, commonly known as Andean climbing catfish, exhibit a unique challenge in species delimitation, leading to ongoing taxonomic debates. Here we report data on Astroblepus mindoensis, a vulnerable species endemic to Ecuador, obtained by an integrative approach that includes cytogenetic analysis, molecular identification of the specimens, and recording of morphological and morphometric characters useful for species diagnosis. Thus, this study aimed to associate the karyotype data of the specimens analyzed with morphological and molecular characters, improving and expanding the existing taxonomic information, thus contributing to the systematics of the species. Our morphology results, unlike Regan's original description, which is brief and ambiguous, provide a more detailed morphometric and meristic description. Molecular phylogenetic reconstruction and genetic distance based on a fragment of the cytochrome c oxidase subunit I (COI) showed that our samples constitute a well‐supported and monophyletic clade within the A. grixalvii species complex. The cytogenetic analysis identified distinct chromosomal markers, including a single cluster of major ribosomal genes (on chromosome pair 3) and of minor ribosomal genes (on chromosome pair 12) with their localization differing from those reported in other Astroblepus species analyzed. Additionally, the presence of a heteromorphic chromosome pair in males suggests the presence of an XX/XY sex‐determination system that has not been identified in other congeneric species. Further investigation is necessary to determine if these chromosomes are associated with the accumulation of repeated sequences, as typically occurs with sex chromosomes, and to assess their presence in other species of the genus. [ABSTRACT FROM AUTHOR]

The animal gut microbiome can have a strong influence on the health, fitness, and behavior of its hosts. The composition of the gut microbial community can be influenced by factors such as diet, environment, and evolutionary history (phylosymbiosis). However, the relative influence of these factors is unknown in most bird species. Furthermore, phylosymbiosis studies have largely focused on clades that diverged tens of millions of years ago, and little is known about the degree of gut microbiome divergence in more recent species radiations. This study explores the drivers of microbiome variation across the unique and recent Hawaiian honeycreeper radiation (Fringillidae: Drepanidinae). Fecal samples were collected from 14 extant species spanning the main islands of the Hawaiian archipelago and were sequenced using three metabarcoding markers to characterize the gut microbiome, invertebrate diet, and plant diet of Hawaiian honeycreepers. We then used these metabarcoding data and the honeycreeper host phylogeny to evaluate their relative roles in shaping the gut microbiome. Microbiome variation across birds was highly individualized; however, source island had a small but significant effect on microbiome structure. The microbiomes did not recapitulate the host phylogenetic tree, indicating that evolutionary history does not strongly influence microbiome structure in the honeycreeper clade. These results expand our understanding of the roles of diet, geography, and phylogeny on avian microbiome structure, while also providing important ecological information about the diet and gut microbiota of wild Hawaiian honeycreepers. [ABSTRACT FROM AUTHOR]

Pollination is an ecosystem process that is crucial to maintain biodiversity and ecosystem function. Bats are important pollinators in the tropics and are an integral part of complex plant–pollinator interaction networks. However, network analysis–based approaches are still scarce at the plant species and bat community levels. We used metabarcoding to identify plant taxa present in pollen from fur and faecal samples collected across 1 year from three nectar‐feeding bat roosts in central Mexico. We calculated the frequency of occurrence of plant taxa and assembled a zoocentric network of bat–plant interactions. We constructed a year‐long network, encompassing the entire period of sampling, two seasonal networks comprising the wet and dry seasons, and six individual networks from sampling at two‐month intervals across the year. Four species of nectar‐feeding bats interacted with 36 plant species from 16 families. We found highly generalised interaction patterns across networks corresponding with opportunistic feeding behaviour by bats, with little seasonal variation in network structure. There was high resource overlap between bat species, and bats visited a diverse range of plant species even during periods with a high abundance of particular resources in the landscape. The diverse diet of nectar‐feeding bats emphasises the importance of floristically rich natural habitats in the landscape to provide reliable foraging resources year‐round in a seasonally variable system. While a generalised network structure is thought to increase robustness, further research is necessary to understand how fluctuations in pollinator abundance and diversity in the face of land use and climate change may impact bat–flower networks and the consequences to plant communities. [ABSTRACT FROM AUTHOR]

The Ili River Valley, located in the northwest of China, serves as a vital repository for fish genetic resources. Its extensive water network and diverse climate have given rise to a unique fish composition and endemic species. In this study, we collected the cytochrome c oxidase subunit I (COI) sequences from 660 fish specimens in the Ili River Valley. The effectiveness of DNA barcoding in identifying fish species in the area was assessed by examining genetic distances, constructing phylogenetic trees, and performing ABGD (Automatic Barcode Gap Discovery) analyses, among other methods. In total, 20 species were identified, including one unidentified species (Silurus sp.). Except for Silurus asotus and Hypophthalmichthys molitrix (only one sample), the maximum intraspecific genetic distance among the remaining species was smaller than the minimum interspecific distance, which proves that the species exhibit obvious barcode gaps. In the Neighbor‐Joining trees, 20 species formed separate monophyletic branches. According to ABGD analysis, 660 sequences were categorized into 19 Operational Taxonomic Units, with Silurus sp. and S. asotus grouped into a single OTU. The Silurus in this study exhibits shared haplotypes and significant genetic divergence, suggesting the potential presence of cryptic species. Furthermore, the nucleotide diversity across all species fell below the threshold level, indicating that the local fish population is gradually declining. In conclusion, this study has demonstrated the effectiveness of DNA barcoding in identifying fish species in the Ili River Valley, providing valuable data to support the conservation of local fish resources. [ABSTRACT FROM AUTHOR]

Phytoplankton have short generation times, flexible reproduction strategies, large population sizes and high standing genetic diversity, traits that should facilitate rapid evolution under directional selection. We quantified local adaptation of copper tolerance in a population of the diatom Skeletonema marinoi from a mining‐exposed inlet in the Baltic Sea and in a non‐exposed population 100 km away. We hypothesized that mining pollution has driven evolution of elevated copper tolerance in the impacted population of S. marinoi. Assays of 58 strains originating from sediment resting stages revealed no difference in the average tolerance to copper between the two populations. However, variation within populations was greater at the mining site, with three strains displaying hyper‐tolerant phenotypes. In an artificial evolution experiment, we used a novel intraspecific metabarcoding locus to track selection and quantify fitness of all 58 strains during co‐cultivation in one control and one toxic copper treatment. As expected, the hyper‐tolerant strains enabled rapid evolution of copper tolerance in the mining‐exposed population through selection on available strain diversity. Within 42 days, in each experimental replicate a single strain dominated (30%–99% abundance) but different strains dominated the different treatments. The reference population developed tolerance beyond expectations primarily due to slowly developing plastic response in one strain, suggesting that different modes of copper tolerance are present in the two populations. Our findings provide novel empirical evidence that standing genetic diversity of phytoplankton resting stage allows populations to evolve rapidly (20–50 generations) and flexibly on timescales relevant for seasonal bloom progressions. [ABSTRACT FROM AUTHOR]

Spoilage of postharvest strawberries by fungal pathogens is a major global concern. This study employed culture‐dependent and culture‐independent methods to identify postharvest fungal communities of strawberries in Western Australia (WA). Ripe strawberries from eight varieties were sampled twice, early and late in the growing season, from 20 farms in two growing regions. At the first sampling time, traditional isolation and identification demonstrated a high abundance of Botrytis and Cladosporium in Region 1. Mucor was abundant at the second sampling time in Region 1 and both times in Region 2. Metabarcoding confirmed variety‐specific fungal communities in WA strawberries. Botrytis and Cladosporium were predominant in Region 1 and Region 2 samples with more than 50% read abundance except on variety Suncoast. No Mucor sequences were detected using metabarcoding, although they were isolated from all samples using culture‐based methods. Sisquoc, a hydroponic variety, had a significantly higher fungal diversity than the soil‐grown varieties. Varieties Suncoast and Albion exhibited the lowest fungal diversity compared to others. This study demonstrated the value of using culture‐dependent and culture‐independent methods to investigate the complexity of fungal microbiomes of postharvest strawberries. [ABSTRACT FROM AUTHOR]

Describing living community compositions is essential to monitor ecosystems in a rapidly changing world, but it is challenging to produce fast and accurate depiction of ecosystems due to methodological limitations. Morphological methods provide absolute abundances with limited throughput, whereas metabarcoding provides relative abundances of genes that may not correctly represent living communities from environmental DNA assessed with morphological methods. However, it has the potential to deliver fast descriptions of living communities provided that it is interpreted with validated species‐specific calibrations and reference databases. Here, we developed a quantitative approach to retrieve from metabarcoding data the assemblages of living large benthic foraminifera (LBF), photosymbiotic calcifying protists, from Indonesian coral reefs that are under increasing anthropogenic pressure. To depict the diversity, we calculated taxon‐specific correction factors to reduce biological biases by comparing surface area, biovolume and calcite volume, and the number of mitochondrial gene copies in seven common LBF species. To validate the approach, we compared calibrated datasets of morphological communities from mock samples with bulk reef sediment; both sample types were metabarcoded. The calibration of the data significantly improved the estimations of genus relative abundance, with a difference of ±5% on average, allowing for comparison of past morphological datasets with future molecular ones. Our results also highlight the application of our quantitative approach to support reef monitoring operations by capturing fine‐scale processes, such as seasonal and pollution‐driven dynamics, that require high‐throughput sampling treatment. [ABSTRACT FROM AUTHOR]

Molecular techniques like metabarcoding, while promising for exploring diversity of communities, are often impeded by the lack of reference DNA sequences available for taxonomic annotation. Our study explores the benefits of combining targeted DNA barcoding and morphological taxonomy to improve metabarcoding efficiency, using beach meiofauna as a case study. Beaches are globally important ecosystems and are inhabited by meiofauna, microscopic animals living in the interstitial space between the sand grains, which play a key role in coastal biodiversity and ecosystem dynamics. However, research on meiofauna faces challenges due to limited taxonomic expertise and sparse sampling. We generated 775 new cytochrome c oxidase I DNA barcodes from meiofauna specimens collected along the Netherlands' west coast and combined them with the NCBI GenBank database. We analysed alpha and beta diversity in 561 metabarcoding samples from 24 North Sea beaches, a region extensively studied for meiofauna, using both the enriched reference database and the NCBI database without the additional reference barcodes. Our results show a 2.5‐fold increase in sequence annotation and a doubling of species‐level Operational Taxonomic Units (OTUs) identification when annotating the metabarcoding data with the enhanced database. Additionally, our analyses revealed a bell‐shaped curve of OTU richness across the intertidal zone, aligning more closely with morphological analysis patterns, and more defined community dissimilarity patterns between supralittoral and intertidal sites. Our research highlights the importance of expanding molecular reference databases and combining morphological taxonomy with molecular techniques for biodiversity assessments, ultimately improving our understanding of coastal ecosystems. [ABSTRACT FROM AUTHOR]

Sciadonus alphacrucis Melo, Gomes, Møller & Nielsen, 2022 is a rare deep‐sea species, previously known from only two specimens collected off São Paulo State, southeastern Brazil, in the western South Atlantic. Herein, we report a new specimen of S. alphacrucis collected on the continental slope off Santa Catarina State, southern Brazil, thereby extending its known distribution by 420 km. Additionally, we provide the new meristic and morphometric data, the molecular identification using sequences of the cytochrome c oxidase subunit I (COI), an updated distribution map, and a discussion of troglomorphic traits. [ABSTRACT FROM AUTHOR]

A new, distinctively short‐bodied giraffe catfish of Parauchenoglanis is described from the Ndzaa River, a small left‐bank tributary of the Mfimi‐Lukenie basin in the Central basin of the Congo River in the Democratic Republic of the Congo. The new species can be distinguished from all congeners by having 29 or fewer (vs. 33 or more) total vertebrae. It can further be distinguished from all congeners, except Parauchenoglanis zebratus Sithole et al., 2023 and Parauchenoglanis ngamensis (Boulenger 1911), by having 13 or 14 (vs. 16 or more) pre‐anal vertebrae. The species is endemic to the Mfimi River basin, where it has been collected mainly in blackwater tributaries. [ABSTRACT FROM AUTHOR]

Barathronus is a genus of blind cusk eels comprising 11 valid species. In this paper, we report the second specimen ever documented of Barathronus roulei (Bythitidae) obtained from the Porcupine Bank by R.V. Vizconde de Eza using a bottom trawl at a depth of 1349 m. Morphological description and illustrations, including a radiograph, are provided. In addition, three new sequences corresponding to three different genes, cytochrome c oxidase subunit I (COI)‐DNA barcoding, 16S ribosomal RNA (16S), and recombination activating protein 1 (RAG1), have been added to the molecular repositories, representing the first sequences for the species. [ABSTRACT FROM AUTHOR]

Neogoniolithon is a widespread genus of non‐geniculate coralline algae in the Mediterranean Sea. Three vaguely differentiated species have been reported in the region. This study examined the diversity of Neogoniolithon in the Mediterranean by inferring phylogenies using psbA and COI‐5P markers, applying delimitation algorithms and comparing morpho‐anatomies of putative species. In contrast to previous reports, 13 species were delimited from the Mediterranean in two distant clades. Initial observations suggested that tetrasporangial conceptacle size might be used to separate the two clades. Only three species showed fruticose morphologies. Further morpho‐anatomical differentiation was difficult. One species included Mediterranean, Pacific and Atlantic samples, while three other species occurred in the Mediterranean and the Canary Islands. The global tree revealed that most species were geographically restricted. Crypticity in multiple lineages implied a much higher diversity than currently reported, even lacking samples from the eastern Mediterranean. Only sequence data can be used to identify these cryptic lineages. High endemism emphasizes the importance of conservation of coralline algal bioconstructions to prevent extinctions. The presence of cryptic diversity also invites a reassessment of the knowledge on species distributions that are important in conservation planning and management. [ABSTRACT FROM AUTHOR]

North American river otters (Lontra canadensis) are semiaquatic mammals distributed throughout the North American continent. They are generalist carnivores with a high potential to affect community structure and dynamics. However, habitat degradation and ecosystem changes have the potential to impact their trophic interactions. Therefore, investigating their diet is critical to understand how anthropogenic disturbances can affect their ecological roles, particularly in the urbanised ecosystems in Florida, where such diet information is currently unavailable. Due to their elusive nature, direct observation of feeding events is a major challenge. Here, we have applied DNA metabarcoding of faecal matter (fDNA), opportunistically collected from river otters killed by road traffic in eastern Florida. Two mitochondrial markers were used to identify prey species' DNA—one for fish and one for vertebrates. A total of 23 unique exact sequence variants (ESVs) were identified, the majority belonging not only to teleosts but also to amphibians, reptiles, birds and mammals. The prey species identified are known dietary resources for river otters and the recovered dietary profiles provided independently by the fish and vertebrate assays were similar. Among the identified teleost prey, three fish species are considered invasive. Here, we show that when sufficient reference sequences are available, fDNA metabarcoding provides high prey species resolution. The collection and metabarcoding of faecal samples is particularly advantageous for scaling diet studies, both geographically and longitudinally, highlighting the potential of fDNA metabarcoding for the investigation of the diet of predators. This can provide foundational information on the organisation of ecological networks, which is vital for implementing successful ecosystem‐based conservation management. [ABSTRACT FROM AUTHOR]

DNA barcodes (i.e., gene sequences used as identifiers for species identification) constitute a very useful tool in invasive pest research. Using them, we studied, for the first time, the origin and population structure of the alien Cydia pomonella in Africa, a major apple pest. We sequenced a fragment of the mitochondrial gene Cytochrome c oxidase subunit I (COI) from C. pomonella caterpillars collected in northern Algeria and compared them with DNA barcodes from the rest of the world available at public repositories. The phylogeny built upon that COI database supports the European origin of the species: haplotypes at the base of the phylogeny were registered in this continent. In Algeria, the results suggest that this pest could have been introduced from Europe, with two unique African mutations probably favoured by C. pomonella multivoltinism and large population sizes. Population genetic analyses at three Algerian localities showed neither structure nor founder effects. However, to confirm the underlying process of the observed population patterns, it is necessary to perform analyses using genes with higher mutation rates. Compared to higher resolution markers, mitochondrial DNA barcodes are still a cost‐effective tool for taxonomic determination of unknown specimens and, sometimes, may also allow a preliminary tracing of its origin. This is crucial to detect and stop the spread of invasive pests (i.e., borders). We encourage further studies including functional genes to assess whether any mutations are promoting C. pomonella adaptation to the Northern Africa environment. [ABSTRACT FROM AUTHOR]

Identification of fish larvae based on morphology is typically limited to higher taxonomic ranks (e.g., family or order), as larvae possess few morphological diagnostic characters for precise discrimination to species. When many samples are presented at any one time, the use of morphology to identify such specimens can be laborious and time‐consuming. Using a reverse workflow for specimen sorting and identification leveraging high‐throughput DNA sequencing, thousands of fish larvae can be DNA barcoded and sorted into molecular operational taxonomic units (mOTUs) in a single sequencing run with the nanopore sequencing technology (e.g., MinION). This process reduces the time and financial costs of morphology‐based sorting and instead deploys experienced taxonomists for species taxonomic work where they are needed most. In this study, a total of 3022 fish larval specimens from plankton tows across four sites in Singapore were collected and sorted based on this workflow. Eye tissue from individual samples was used for DNA extraction and sequencing of cytochrome c oxidase subunit I. We generated a total of 2746 barcodes after quality filtering (90.9% barcoding success), identified 2067 DNA barcodes (75.3% identification success), and delimited 256 mOTUs (146 genera, 52 families). Our analyses identified specific challenges to species assignment, such as the potential misidentification of publicly available sequences used as reference barcodes. We highlighted how the conservative application and comparison of a local sequence database can help resolve identification conflicts. Overall, this proposed approach enables and expedites taxonomic identification of fish larvae, contributing to the enhancement of reference barcode databases and potentially better understanding of fish connectivity. [ABSTRACT FROM AUTHOR]

Premise: Sphagnum magellanicum (Sphagnaceae, Bryophyta) has been considered to be a single semi‐cosmopolitan species, but recent molecular analyses have shown that it comprises a complex of at least seven reciprocally monophyletic groups, that are difficult or impossible to distinguish morphologically. Methods: Newly developed barcode markers and RADseq analyses were used to identify species among 808 samples from 119 sites. Molecular approaches were used to assess the geographic ranges of four North American species, the frequency at which they occur sympatrically, and ecological differentiation among them. Microhabitats were classified with regard to hydrology and shade. Hierarchical modelling of species communities was used to assess climate variation among the species. Climate niches were projected back to 22,000 years BP to assess the likelihood that the North American species had sympatric ranges during the late Pleistocene. Results: The species exhibited parallel morphological variation, making them extremely difficult to distinguish phenotypically. Two to three species frequently co‐occurred within peatlands. They had broadly overlapping microhabitat and climate niches. Barcode‐ versus RADseq‐based identifications were in conflict for 6% of the samples and always involved S. diabolicum vs. S. magniae. Conclusions: These species co‐occur within peatlands at scales that could permit interbreeding, yet they remain largely distinct genetically and phylogenetically. The four cryptic species exhibited distinct geographic and ecological patterns. Conflicting identifications from barcode vs. RADseq analyses for S. diabolicum versus S. magniae could reflect incomplete speciation or hybridization. They comprise a valuable study system for additional work on climate adaptation. [ABSTRACT FROM AUTHOR]

Research Highlight: Bourbour et al., 2024. Feeding en route: Prey availability and traits influence prey selection by an avian predator on migration. Journal of Animal Ecology. Diet selection plays a key role in the eco‐evolutionary dynamics of animals, exhibiting substantial variability across species, environments and seasons. The complex interplay between movement capability, hunting strategies, habitat use, prey traits and availability shapes the foraging patterns of avian predators. However, detailed information on how these birds exploit their extensive territories remains limited. In this study, Bourbour et al. utilised a novel integration of eDNA metabarcoding and citizen science to explore predator–prey interactions between migrating sharp‐shinned hawks (Accipiter striatus) and an ephemeral avian prey community along North America's Pacific flyway. The research identified 1396 detections from the diet (65 species) of 588 migrating sharp‐shinned hawks. Hawks' diet composition correlated with prey abundance indices sourced from the eBird database throughout the migration season, highlighting the significant impact of prey availability—shaped by migration tendency, flocking behaviour, and habitat—on raptor‐songbird interactions. Notably, the study also found significant differences in prey size between male and female hawks, indicating that sexual dimorphism has led to diverse foraging strategies during migration. These findings underscore the potential of combining eDNA metabarcoding with citizen science to deepen our understanding of the foraging ecology of highly mobile and wide‐ranging birds, as well as to monitor complex and vast ecosystems. [ABSTRACT FROM AUTHOR]

Premise: Plants and pollinators closely interact with each other to form complex networks of species interactions. Metabarcoding of pollen collections has recently been proposed as an advantageous method for the construction of such networks, but the extent to which diversity and community analyses depend on the extraction method and pollen concentration used remains unclear. Methods: In this study, we used a dilution series of two pollen mixtures (a mock community and pooled natural pollen loads from bumblebees) to assess the effect of mechanical homogenization and two DNA extraction kits (spin column DNA extraction kit and magnetic bead DNA extraction kit) on the detected pollen richness and community composition. Results: All species were successfully detected using the three methods, even in the most dilute samples. However, the extraction method had a significant effect on the detected pollen richness and community composition, with simple mechanical homogenization introducing an extraction bias. Discussion: Our findings suggest that all three methods are effective for detecting plant species in the pollen loads on insects, even in cases of very low pollen loads. However, our results also indicate that extraction methods can have a profound impact on the ability to correctly assess the community composition of the pollen loads on insects. The choice of extraction methodology should therefore be carefully considered to ensure reliable and unbiased results in pollen diversity and community analyses. [ABSTRACT FROM AUTHOR]

An accurate species‐level taxonomy is paramount for biogeographical research, and conversely, biogeographical data are of importance for species delimitation. We here review recent developments and future perspectives of taxonomy of direct relevance for biogeographers. The understanding that species are independently evolving segments of population‐level lineages, the rise of integrative approaches to delimit such lineages, and the advent of high‐throughput sequencing have considerably renewed the discipline of taxonomy. Using genome‐scale molecular datasets, the extent of admixture across hybrid zones can now be effectively assessed and the evolutionary independence of lineages inferred, leading to more reliable and comparable species delimitation criteria. Substantially divergent but admixing phylogeographical lineages can conveniently be named as subspecies, thus avoiding taxonomic oversplitting and taxonomic inflation. At the same time, comprehensive DNA barcoding and DNA metabarcoding efforts are uncovering an enormous proportion of undiscovered biotic diversity, and we encourage the development of bioinformatic pipelines that combine high‐throughput species discovery and delimitation with diagnosis and scientific naming, to approach a biotic inventory of the globe without abandoning the established Linnaean system. [ABSTRACT FROM AUTHOR]

Mansonia mosquito species are recognised as a significant vector of human pathogens, primarily transmitting the filarial nematode, Brugia malayi. In central Thailand, the three most prevalent Mansonia species are Mansonia annulifera, Mansonia indiana and Mansonia uniformis. This study explored the influence of seasonal changes on the phenotypic variation of these Mansonia species in central Thailand using the geometric morphometrics (GM). To ensure accurate species identification, we integrated GM techniques with DNA barcoding, examining distinctions in both phenotype and genotype among the species. The intraspecific genetic divergence ranged from 0.00% to 1.69%, whereas the interspecific genetic divergence ranged from 10.52% to 16.36%. The clear distinction between intra‐ and interspecific distances demonstrated the presence of a barcoding gap, confirming the successful differentiation of the three Mansonia mosquito species through DNA barcoding. Similarly, the interspecies GM assessment for classifying Mansonia species demonstrated a high degree of accuracy, with an overall performance of 98.12%. Exploring seasonal variation in the three Mansonia species revealed wing variations across different seasons, and pronounced variations appearing in the cool season. Regarding their association with meteorological factors, Ma. annulifera and Ma. uniformis showed significant positive correlations with temperature (p < 0.05), and Ma. uniformis also displayed a significant negative correlation with atmospheric pressure (p < 0.05). The insights from this study will deepen our understanding of the adaptive patterns of Mansonia mosquitoes in Thailand's central region, paving the way for enhanced disease surveillance related to these vectors. [ABSTRACT FROM AUTHOR]

This work demonstrates for the first time using molecular markers alongside chromosomes and the intermediate phenotype that a specimen of Dorcadion is an accidental first‐generation hybrid. The analyses were based on two mitochondrial and five nuclear molecular markers. While analysing the putative parental taxa of the hybrid, it was revealed that even Dorcadion lugubre lugubre Kraatz is likely a hybridogenic taxon. Coincidentally, a new lead was discovered concerning the parental taxa of Dorcadion aethiops (Scopoli), already known as a hybridogenic species. This new evidence of hybridisation adds to the recent publications to strengthen the hypothesis that, in Dorcadionini, this phenomenon is relatively frequent, and speciation is often reticulate: New taxa can appear through introgressive hybridisation, explaining in part the extreme species richness of the group. [ABSTRACT FROM AUTHOR]

Caloptilia roscipennella (Hübner, 1796) is a leaf‐mining moth native to Central and Southern Europe. In this publication, we provide the first report of the occurrence of Caloptilia roscipennella in India. The identification was performed using an integrative approach, combining molecular (barcoding, COI) and morphological analysis (forewing pattern). In 2020, the larvae were initially found scraping in the silvery‐white mine and later feeding in folded or rolled leaves of the common walnut Juglans regia L. In the subsequent years, the species was found only in the same places. Two generations were recorded per year (May–June and August–September) and leaf miners overwintered as adults and reappeared in the early spring. Damage ranging from 15% to 20% was observed on the leaves of J. regia. Further efforts related to C. roscipennella should concentrate on evaluating its geographic distribution and its impact on walnut trees. Several species of parasitoids were reared from infested mines, with specimens belonging to the genus Chrysocharis Förster, 1856 (Hymenoptera: Eulophidae) and Itoplectis maculator (Fabricius, 1775) (Hymenoptera: Ichneumonidae). These species provided excellent control of leaf miner larvae, and out of 10 larvae, seven were found parasitized by the parasitoids and higher numbers of specimens were found with relative higher frequencies. [ABSTRACT FROM AUTHOR]

Gut content metabarcoding has provided important insights into the food web ecology of spiders, the most dominant terrestrial arthropod predators. In small invertebrates, like spiders, gut content analysis is often performed on whole body DNA extracts of individual predators, from which prey sequences are selectively enriched and sequenced.Since many spider species are generalist predators, large numbers of samples comprising individual spider specimens must be analysed to recover an exhaustive image of a spider species' prey spectrum, which is costly and time‐consuming.Pooled processing of bulk samples of multiple specimens has been suggested to reduce the necessary workload and cost while still recovering a representative estimate of the prey diversity. However, it is still unclear if pooling approaches lead to bias in recovering the prey spectrum and if the results are comparable with data from individually processed spiders.Here, we test the effects of metabarcoding pooled spider gut content on the recovered taxonomic diversity and composition of prey. Using a newly adapted primer pair, which efficiently enriches COI barcode sequences of diverse arthropod prey groups while suppressing spider amplification, we test if pooling leads to reduced taxonomic diversity or skewed estimates of prey composition.Our results show that pooling and individual processing recover highly correlated taxonomic diversity and composition of prey. The only exception are very rare prey items which were less well recovered by pooling. Our results support pooling as a cost‐effective and time‐efficient approach to recover the diet of generalist predators for population‐level studies of spider trophic interactions. [ABSTRACT FROM AUTHOR]

BACKGROUND: Crustaceans of the superfamily Penaeoidea (e.g., shrimps and prawns) are among the most commercially available aquatic products worldwide. However, there are few studies regarding not only the presence but also the characteristics of mislabelling in these food products. Such information would be helpful for consumers in order to avoid the typical problems associated with mislabelling (e.g., health and economic issues). For this reason, this work considers Penaeoidea mislabelling by comparing different products (frozen, fresh, boiled), and sources (hypermarkets, supermarkets and fishmongers) from Spain (Europe). RESULTS: A total of 94 samples from 55 different products were collected, representing 19 different species from 13 genera. Mitochondrial DNA (COI gene) was amplified, revealing mislabelling in almost 30% of supermarket products and almost exclusively found in frozen samples (95% of the total) regardless of its price. In addition, products from the Pacific Ocean seem to be particularly susceptible to mislabelling. CONCLUSIONS: All in all, recommendations for the consumer in order to avoid mislabelling of prawns include purchasing them fresh from fishmongers; aquaculture products must not be avoided. This study represents, to our knowledge, the first attempt to provide recommendations to consumers based on DNA analyses in order to avoid mislabelling in food products. Further research is therefore required to provide such recommendations in different food products, particularly those that are processed, packaged and/or frozen. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

ABSTRACT The first record of the kelp species Phyllariopsis brevipes in the Campania Region (Tyrrhenian Sea, South Italy) is here reported. The species has been observed in October 2021 in four sites along Sorrento Peninsula and Capri Island coast during scuba diving surveys. P. brevipes grew on living thalli of crustose coralline algae from 32 up to 55 m depth, occasionally forming dense aggregations with a maximum cover of about 0.2 m2. The DNA barcoding analysis on the basis of the cox1 gene supported the identification, with 98.36% of similarity between the sample collected in this study (NCBI accession number: PP622796) and a sequence obtained from a specimen from Provence, Mediterranean France (NCBI accession number: GQ368264). The study area can be considered a stepping stone for the species dispersion. The occurrence of P. brevipes can be related to upwelling and good water quality and the monitoring of its health status might be a sentinel of environmental changes. [ABSTRACT FROM AUTHOR]

Aotearoa (New Zealand) is a biodiversity hotspot for temperate invertebrate taxa and home to high levels of endemicity. However, our knowledge of species‐level diversity and phylogeny of endemic New Zealand Onychophora (velvet worms) is at present limited. Here, we use mitochondrial cytochrome c oxidase subunit I (COI) barcoding to assess the extent of species diversity for the two velvet worm genera found in New Zealand, the ovoviviparous and endemic Peripatoides and the oviparous Ooperipatellus, found in Australia and New Zealand. Our results reveal that the estimated number of species of both genera in New Zealand is greater than currently described. We estimate there are between 13 and 67 species of Peripatoides and between 16 and 21 species of Ooperipatellus endemic to New Zealand. This is a stark increase from the two currently described New Zealand species of Ooperipatellus and previous work that has identified 10 species within Peripatoides. Our exploration of climatic variables shows that individuals of Ooperipatellus are predominantly found in wet, cool environments and Peripatoides are found across relatively drier, warmer habitats. We also generate ecological niche models to provide initial predictions of the distribution of climatically suitable habitats for each genus across New Zealand. [ABSTRACT FROM AUTHOR]

Multispecies and ecosystem models, which are key for the implementation of ecosystem‐based approaches to fisheries management, require extensive data on the trophic interactions between marine organisms, including changes over time. DNA metabarcoding, by allowing the simultaneous taxonomic identification of the community present in hundreds of samples, could be used for speeding up large‐scale stomach content data collection. Yet, for DNA metabarcoding to be routinely implemented, technical challenges should be addressed, such as the potentially complicated sampling logistics, the detection of a high proportion of predator DNA, and the inability to provide reliable abundance estimations. Here, we present a DNA metabarcoding assay developed to examine the diet of five commercially important fish, which can be feasibly incorporated into routinary samplings. The method is devised to speed up the analysis process by avoiding the stomach dissection and content extraction steps, while preventing the amplification of predator DNA by using blocking primers. Tested in mock samples and in real stomach samples, the method has proven effective and shows great effectiveness discerning diet variations due to predator ecology or prey availability. Additionally, by applying our protocol to mackerel stomachs previously analyzed by visual inspection, we showcase how DNA metabarcoding could complement visually based data by detecting overlooked prey by the visual approach. We finally discuss how DNA metabarcoding‐based data can contribute to trophic data collection. Our work reinforces the potential of DNA metabarcoding for the study and monitoring of fish trophic interactions and provides a basis for its incorporation into routine monitoring programs, which will be critical for the implementation of ecosystem‐based approaches to fisheries management. [ABSTRACT FROM AUTHOR]

The Pinna nobilis, a Mediterranean mollusc, has suffered population declines due to a massive mortality event associated with various factors including the parasite Haplosporidium pinnae. Some populations show resilience, possibly due to local environmental conditions. In this study, a molecular multiplex PCR method was developed using species‐specific primers targeting Internal Transcribed Spacer (ITS) regions of P. nobilis and P. rudis, allowing accurate species identification and hybrid detection. Samples from Mediterranean areas were analysed, including putative hybrids and individuals from five other bivalve species. DNA was isolated, ITS regions were amplified and sequenced, and phylogenetic analyses confirmed species differentiation and primer specificity. The multiplex‐PCR successfully identified P. nobilis, P. rudis, and their hybrids based on distinct amplicon patterns. This study highlights the value of molecular tools in species conservation, especially for monitoring and managing hybridization, supporting effective biodiversity conservation strategies. [ABSTRACT FROM AUTHOR]

Rivers are crucial ecosystems supporting biodiversity and human well‐being, yet they face increasing degradation globally. Traditional river biomonitoring methods based on morphological identification of macroinvertebrates present challenges in terms of taxonomic resolution and scalability. This study explores the application of DNA metabarcoding analysis in both bulk and environmental DNA (eDNA) samples for comprehensive assessment of macrozoobenthic biodiversity, detection of invasive and endangered species, and evaluation of river ecological status in northwestern Spain. DNA metabarcoding of homogenized bulk samples and water eDNA revealed a mean of 100 and 87 macrozoobenthos species per sample respectively. However, the specific composition was significantly different with only 27.3% of the total species being shared. It was not possible to identify all the OTUs to species level; only 17.43% and 49.4% of the OTUs generated could be identified to species level in the bulk and eDNA samples, respectively. Additionally, a total of 11 exotic species (two first records for the Iberian Peninsula and another three first records for Asturias region) and one endangered species were detected by molecular tools. Molecular methods showed significant correlations with morphological identification for EQR values (Ecological Quality Ratio) of IBMWP index, yet differences in inferred river ecological status were noted, with bulk samples tending to indicate higher status. Overall, DNA metabarcoding offers a promising approach for river biomonitoring, providing insights into biodiversity, invasive species, and ecological status within a single analysis. Further optimization and intercalibration are required for its implementation in routine biomonitoring programmes, but its scalability and multi‐tasking capabilities position it as a valuable tool for integrated monitoring of river ecosystems. [ABSTRACT FROM AUTHOR]

Biogeographic structure in marine protist communities is shaped by a combination of dispersal potential and environmental selection. High‐throughput sequencing and global sampling efforts have helped better resolve the composition and functions of these communities in the world's oceans using both molecular and visual methods. However, molecular barcoding data are critically lacking across the Indo‐Pacific, a region widely considered the epicenter of marine biodiversity. To fill this gap, we characterized protist communities in four sampling regions across Indonesia that represent the latitudinal, longitudinal, and human population gradients of the region: Lombok, Wakatobi, Misool, and Waigeo. We show high spatial structuring in marine protist communities across Indonesia, and biotic factors appear to play little role in driving this observed structure. Our results appear to be driven by abiotic factors linked to surface current patterns across the Indo‐Pacific as a result of: (1) a choke point in circulation at the Indonesian Throughflow leading to low diatom diversity in Lombok, Wakatobi, and Misool; (2) an increase in nutrient availability at the edge of the Halmahera Eddy in Waigeo, leading to an increase in diatom diversity; and/or (3) seasonal variations in protist communities in line with shifts in velocity of the Indonesian Throughflow. Overall, our results highlight the importance of abiotic factors in shaping protist communities on broad geographic scales over biotic, top‐down pressures, such as grazing from higher trophic levels. [ABSTRACT FROM AUTHOR]

Phytoplankton and bacteria form the foundation of marine food webs. While most studies on phytoplankton bloom influence on bacteria dynamics focus on diatom‐dominated blooms due to their global ecological significance, it is unclear if similar patterns extend to other species that compete with diatoms like Phaeocystis spp. This study aimed to contribute to the understanding of associations between phytoplankton and bacteria in a temperate ecosystem. For this, we studied the dynamics of phytoplankton and bacteria, combining 16S metabarcoding, microscopy, and flow cytometry over 4 years (282 samples). Phytoplankton and bacterial communities were studied throughout the year, particularly during contrasting phytoplankton blooms dominated by the Haptophyte Phaeocystis globosa or diatoms. We applied extended local similarity analysis (eLSA) to construct networks during blooming and non‐blooming periods. Overall, the importance of seasonal and species‐specific interactions between phytoplankton and bacteria is highlighted. In winter, mixed diatom communities were interconnected with bacteria, indicating a synergistic degradation of diverse phytoplankton‐derived substrates. In spring, despite the intensity variations of P. globosa blooms, the composition of bacterial communities remained consistent over several years, suggesting establishing a stable‐state environment for bacterial communities. Specific associations between monospecific diatom blooms and bacteria were evidenced in summer. [ABSTRACT FROM AUTHOR]

The utility of a universal DNA 'barcode' fragment (658 base pairs of the Cytochrome C Oxidase I [COI] gene) has been established as a useful tool for species identification, and widely criticized as one for understanding the evolutionary history of a group. Large amounts of COI sequence data have been produced that hold promise for rapid species identification, for example, for biosecurity. The fruit fly tribe Dacini holds about a thousand species, of which 80 are pests of economic concern. We generated a COI reference library for 265 species of Dacini containing 5601 sequences that span most of the COI gene using circular consensus sequencing. We compared distance metrics versus monophyly assessments for species identification and although we found a 'soft' barcode gap around 2% pairwise distance, the exceptions to this rule dictate that a monophyly assessment is the only reliable method for species identification. We found that all fragments regularly used for Dacini fruit fly identification >450 base pairs long provide similar resolution. 11.3% of the species in our dataset were non‐monophyletic in a COI tree, which is mostly due to species complexes. We conclude with recommendations for the future generation and use of COI libraries. We revise the generic assignment of Dacus transversus stat. rev. Hardy 1982, and Dacus perpusillus stat. rev. Drew 1971 and we establish Dacus maculipterus White 1998 syn. nov. as a junior synonym of Dacus satanas Liang et al. 1993. [ABSTRACT FROM AUTHOR]

Metabarcoding‐based methods for identification of host‐associated eukaryotes have the potential to revolutionize parasitology and microbial ecology, yet significant technical challenges remain. In particular, highly abundant host reads can mask the presence of less‐abundant target organisms, especially for sample types rich in host DNA (e.g., blood and tissues). Here, we present a new CRISPR‐Cas9‐mediated approach designed to reduce host signal by selective amplicon digestion, thus enriching clinical samples for eukaryotic endosymbiont sequences during metabarcoding. Our method achieves a nearly 76% increased efficiency in host signal reduction compared with no treatment and a nearly 60% increased efficiency in host signal reduction compared with the most commonly used published method. Furthermore, the application of our method to clinical samples allows for the detection of parasite infections that would otherwise have been missed. [ABSTRACT FROM AUTHOR]

DNA barcoding is commonly used for species identification. Despite this, there has not been a comprehensive assessment of the utility of DNA barcoding in crayfishes (Decapoda: Astacidea). Here we examined the extent to which local barcoding gaps (used for species identification) and global barcoding gaps (used for species discovery) exist among crayfishes, and whether global gaps met a previously suggested 10× threshold (mean interspecific difference being 10× larger than mean intra specific difference). We examined barcoding gaps using publicly available mitochondrial COI sequence data from the National Center for Biotechnology Information's nucleotide database. We created two versions of the COI datasets used for downstream analyses: one focused on the number of unique haplotypes (NH) per species, and another that focused on total number of sequences (NS; i.e., including redundant haplotypes) per species. A total of 81 species were included, with 58 species and five genera from the family Cambaridae and 23 species from three genera from the family Parastacidae. Local barcoding gaps were present in only 30 species (20 Cambaridae and 10 Parastacidae species). We detected global barcoding gaps in only four genera (Cambarus, Cherax, Euastacus, and Tenuibranchiurus), which were all below (4.2× to 5.2×) the previously suggested 10× threshold. We propose that a ~5× threshold would be a more appropriate working hypothesis for species discovery. While the NH and NS datasets yielded largely similar results, there were some discrepant inferences. To understand why some species lacked a local barcoding gap, we performed species delimitation analyses for each genus using the NH dataset. These results suggest that current taxonomy in crayfishes may be inadequate for the majority of examined species, and that even species with local barcoding gaps present may be in need of taxonomic revisions. Currently, the utility of DNA barcoding for species identification and discovery in crayfish is quite limited, and caution should be exercised when mitochondrial‐based approaches are used in place of taxonomic expertise. Assessment of the evidence for local and global barcoding gaps is important for understanding the reliability of molecular species identification and discovery, but outcomes are dependent on the current state of taxonomy. As this improves (e.g., via resolving species complexes, possibly elevating some subspecies to the species‐level status, and redressing specimen misidentifications in natural history and other collections), so too will the utility of DNA barcoding. [ABSTRACT FROM AUTHOR]

Niche theory predicts that ecologically similar sympatric species should show differentiation in at least one of the main niche dimensions (time, space, and/or food). Here, we combined observations of breeding timing, nest site selection, and diet (the latter determined using DNA metabarcoding) to analyze the niche overlap and differentiation between two sympatric secondary cavity‐nesting birds, the Japanese Tit Parus minor and the Yellow‐rumped Flycatcher Ficedula zanthopygia. The results showed that (1) there were significant differences in the first egg laying date, length of the egg laying period, incubation date, and hatching date between tits and flycatchers, and the breeding time of flycatchers peaked later (about 30 days) than that of tits; (2) the two species had a large overlap in nest site selection, although the canopy coverage and shrub density of flycatchers were significantly higher than those of tits; and (3) the niche overlap in diet was minimal, with both species heavily relying on Lepidoptera (39.6% and 63.7% for tits and flycatchers, respectively), but with flycatchers consuming significantly higher percentages of Lepidoptera, Diptera, and Coleoptera than tits. The results indicate that these two sympatric secondary cavity‐nesting species have significant niche differentiation in breeding time and diet, but little differentiation in nest site selection. [ABSTRACT FROM AUTHOR]