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Multiple Salmonella enterica serovars and strains have been reported to be able to persist inside the foliar tissue of lettuce (Lactuca sativa L.), potentially resisting washing steps and reaching the consumer. Intraspecies variation of the bacterial pathogen and of the plant host can both significantly affect the outcome of foliar colonization. However, current understanding of the mechanisms underlying this phenomenon is still very limited. In this study, we evaluated the foliar fitness of 14 genetically barcoded S. enterica isolates from 10 different serovars, collected from plant and animal sources. The S. enterica isolates were vacuum-infiltrated individually or in pools into the leaves of three- to four-week-old lettuce plants. To estimate the survival capacity of individual isolates, we enumerated the bacterial populations at 0- and 10- days post-inoculation (DPI) and calculated their net growth. The competition of isolates in the lettuce apoplast was assessed through the determination of the relative abundance change of barcode counts of each isolate within pools during the 10 DPI experimental period. Isolates exhibiting varying apoplast fitness phenotypes were used to evaluate their capacity to grow in metabolites extracted from the lettuce apoplast and to elicit the reactive oxygen species burst immune response. Our study revealed that strains of S. enterica can substantially differ in their ability to survive and compete in a co-inhabited lettuce leaf apoplast. The differential foliar fitness observed among these S. enterica isolates might be explained, in part, by their ability to utilize nutrients available in the apoplast and to evade plant immune responses in this niche.

Eukaryotes are important components of primate gut communities. Despite their role in the diversity and structure of the gut ecosystem, microbiome research has focused on the prokaryotic component of the gut community. While gut bacteria are shaped by host phylogeny and diet, these factors are known to have negligible effects on eukaryotic diversity, which is expected to be modulated by the characteristics of the habitat. We assess the influence of landscape composition and configuration on the eukaryome of black and gold howler monkeys (Alouatta caraya). We collected fecal samples from 10 independent social groups inhabiting small forest fragments or orchards enriched with native tree species and applied an 18S rRNA gene fragment metabarcoding approach to describe their eukaryotic communities. We used generalized linear models to assess the power of landscape metrics in predicting the richness, diversity, evenness, and phylogenetic diversity of the eukaryome. Most communities were dominated by Ascomycota, and it is likely that many of the reads had an environmental origin. Forest cover affected eukaryome richness positively and patch density showed a positive relationship with the Simpson’s diversity and Pielou’s evenness indexes. Howler monkeys living in landscapes with lower habitat coverage had a lower eukaryotic alpha-diversity in the fecal microbiome. [ABSTRACT FROM AUTHOR]

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]

The invasion of phytopathogens impacts the composition and associations of the internal microbial inhabitants. Leaf spot is one of the most devastating diseases in eggplant var. Mattu Gulla which is unique in terms of geographic indication (GI) status. Leaf spot samples (asymptomatic and symptomatic) were collected to characterize the fungal community associated with them using culture-based and next-generation ITS rRNA-based metabarcoding approaches. Both methods showed that Ascomycota and Basidiomycota were the predominant phyla in both groups. In the asymptomatic group, Didymosphaeriaceae, Pleosporaceae, Trichomeriaceae, and Capnodiaceae were the most differentially abundant families. In contrast, Phaeosphaeriaceae, Pleosporaceae, Didymellaceae, Rhynchogastremataceae, and Bulleribasidiaceae were the most differentially abundant families in the symptomatic group. At the genus level, Cladosporium was the most differentially abundant genus in the asymptomatic group. In the symptomatic group Alternaria, Remotididymella, Vishniacozyma, Bulleribasidium, Occultifur, Epicoccum, and Loratospora were the abundant genera. The pathotroph-saprotrophic mode was the most common mode identified in both groups, with an increased abundance in the symptomatic group. Seven fungal families and two genera were identified as common according to the culture-based method and NGS analysis based on ITS rRNA metabarcoding. Our study indicated that the composition of the core microbial community varies with plant health status, and a combination of culturable and next-generation ITS rRNA-based metabarcoding approaches could be a reliable option for obtaining a detailed understanding of plant-associated fungal communities. [ABSTRACT FROM AUTHOR]

Ticks transmit pathogens of veterinary and public health importance. Understanding their diversity is critical as infestations lead to significant economic losses globally. To date, over 90 species across three families have been identified in South Africa. However, the taxonomy of most species has not been resolved due to morphological identification challenges. DNA barcoding through the Barcode of Life Data Systems (BOLD) is therefore a valuable tool for species verifications for biodiversity assessments. This study conducted an analysis of South African tick COI barcodes on BOLD by verifying species on checklists, literature, and other sequence databases. The compiled list represented 97 species, including indigenous (59), endemics (27), introduced (2), invasives (1), and eight that could not be classified. Analyses indicated that 31 species (32%) from 11 genera have verified COI barcodes. These are distributed across all nine provinces with the Eastern Cape having the highest species diversity, followed by Limpopo, with KwaZulu-Natal having the least diversity. Rhipicephalus, Hyalomma, and Argas species had multiple barcode index numbers, suggesting cryptic diversity or unresolved taxonomy. We identified 21 species of veterinary or zoonotic importance from the Argasidae and Ixodidae families that should be prioritised for barcoding. Coordinating studies and defining barcoding targets is necessary to ensure that tick checklists are updated to support decision-making for the control of vector-borne diseases and alien invasives. [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]

The importance of non-human DNA in the forensic field has increased greatly in recent years, together with the type of applications. The molecular species identification of animal and botanical material may be crucial both for wildlife trafficking and crime scene investigation. However, especially for forensic botany, several challenges slow down the implementation of the discipline in the routine. Although the importance of molecular analysis of animal origin samples is widely recognized and the same value is acknowledged to the botanical counterpart, the latter does not find the same degree of application. The availability of molecular methods, especially useful in cases where the material is fragmented, scarce or spoiled preventing the morphological identification, is not well known. This work is intended to reaffirm the relevance of non-human forensic genetics (NHFG), highlighting differences, benefits and pitfalls of the current most common molecular analysis workflow for animal and botanical samples, giving a practical guide. A flowchart describing the analysis paths, divided in three major working areas (inspection and sampling, molecular analysis, data processing and interpretation), is provided. More real casework examples of the utility of non-human evidence in forensic investigations should be shared by the scientific community, especially for plants. Moreover, concrete efforts to encourage initiatives in order to promote quality and standardization in the NHFG field are also needed. [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]

Recent advancements in RNA therapeutics highlight the critical need for precision gene delivery systems that target specific organs and cells. Lipid nanoparticles (LNPs) have emerged as key vectors in delivering mRNA and siRNA, offering protection against enzymatic degradation, enabling targeted delivery and cellular uptake, and facilitating RNA cargo release into the cytosol. This review discusses the development and optimization of organ- and cell-specific LNPs, focusing on their design, mechanisms of action, and therapeutic applications. We explore innovations such as DNA/RNA barcoding, which facilitates high-throughput screening and precise adjustments in formulations. We address major challenges, including improving endosomal escape, minimizing off-target effects, and enhancing delivery efficiencies. Notable clinical trials and recent FDA approvals illustrate the practical applications and future potential of LNP-based RNA therapies. Our findings suggest that while considerable progress has been made, continued research is essential to resolve existing limitations and bridge the gap between preclinical and clinical evaluation of the safety and efficacy of RNA therapeutics. This review highlights the dynamic progress in LNP research. It outlines a roadmap for future advancements in RNA-based precision medicine. [Display omitted] [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]

In the present study, the DNA barcoding of the nematode parasite infecting Ompok bimaculatus and Nemacheilus anguilla fish species was carried out in Barvi Reservoir, Maharashtra. To ascertain the taxonomic status of these nematode parasites, an 18S gene marker was used. Accurate identification of fish parasites is essential to formulate preventive strategies and to study host–environment relations. The present study did barcoding of the nematode parasites of the fishes caught from the Barvi Reservoir using the nuclear 18S rDNA (SSU) sequence. The nuclear 18S rDNA (SSU) was amplified into two overlapping amplicons and sequenced to identify the species based on the sequence similarity with the NCBI GenBank database. The present study sequences (both fragments) showed 98% similarity with the species of Eustrongylides. The average genetic distance value between the present study sample and species of Eustrongylides was 0.003. In the phylogenetic tree also, the sequence was clustered with the species of Eustrongylides with significant bootstrap values. The present study identified the nematode parasite of the fish caught from the Barvi Reservoir, as species of Eustrongylides. The species‐level identification could not be possible due to the insufficient/lack of reference sequences in the database. It indicates the knowledge gap concerning the species‐specific molecular markers for nematode parasites of the fish. [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]

Integrated use of molecular and morphological methods reveals unexpected diversity in the cosmopolitan siphonophore genus Nanomia. Species delimitation analyses based on COI and 16S sequences suggest up to three distinct lineages in addition to the previously accepted Nanomia bijuga (Delle Chiaje, 1844) and N. cara A. Agassiz, 1865. Here, we describe the North Pacific Nanomia septata sp. n., previously confused with both N. cara and N. bijuga, and provide improved morphological characters for the identification of these three Nanomia species. Phylogenetic analyses suggest two additional, hitherto undescribed clades from Japanese and Chinese waters, respectively, but the lack of morphological material prevents describing these putative species. The geographic distribution of molecularly and/or morphologically verified observations confirm a warm circumglobal distribution for N. bijuga and a boreal North Atlantic distribution for N. cara. Interestingly, four distinct lineages occur in the North Pacific, sometimes in close proximity. These contrasting patterns of distribution raise questions about pelagic speciation processes. [ABSTRACT FROM AUTHOR]

AbstractGenus Calvatia belongs to a gasteroid fungi which are commonly known as a puffball mushrooms including around 47 species worldwide. Approximately 25 species of the genus Calvatia have been reported previously in India from various forest regions; however, only 15 species of these are accepted on their current taxonomic status because several of them are synonymized with other genera or species. The present study reports the distribution of Calvatia holothurioides which was collected from Jambughoda Wildlife Sanctuary in Gujarat state, India. The identification was carried out based on the morphological characteristics as well as molecular phylogenetic analysis using nuclear rDNA Internal Transcribed Spacer (ITS) and rDNA LSU gene sequences data. Nucleotide sequences of different gene loci (ITS and LSU) were submitted into the BOLD data system for DNA barcoding. Additionally, this study also provides a literature-based checklist for the present status of Calvatia species reported in India so far. [ABSTRACT FROM AUTHOR]

Tardigrade diversity and distribution are enigmatic in most parts of the globe, and only some European countries can boast of a relatively well-studied water bear fauna. However, even these suffer from the lack of genetic data, which would substantiate faunistic data and make biogeographic comparisons easier. Denmark has never been intensively and systematically researched in this regard, thus a citizen science sampling of cryptogams (mosses, liverworts, and lichens) was launched in spring 2023, aiming at a comprehensive biodiversity survey across this insular country. Nearly 700 samples were selected out of 8.000 sent to NHMD, based on the quality of samples, representativeness of various regions of Denmark, and the type of substrate to allow unravelling of potential ecological associations between tardigrades and cryptogams. Importantly, a large fraction of morphological identifications was backed up by DNA barcode data based on ITS-2 (1001 sequences), and in some cases also on COI (93 sequences) and ITS-1 (22 sequences) molecular markers, which are recognised DNA fragments used in species delimitation. We quadruple the number of known Danish limno-terrestrial tardigrade species (55 spp. reported in this paper vs. 14 spp. reported in literature so far, most of which were contentious due to the insufficient knowledge on tardigrade taxonomy), demonstrating the power of integrative taxonomy. No fewer than nine spp. are new to science. This is the first case where tardigrade fauna of an entire country is examined both from morphological and DNA barcoding data perspective. [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]

Bat boxes can be employed to provide short-term mitigation for the loss of trees with bat roost potential. Traditionally bat box utilisation (by long-tailed bats) has been difficult to confirm in NZ, partly due to a lack of published survey techniques to confirm bat box utilisation when bats are not present at the time of sampling. While environmental deoxyribonucleic acid (eDNA) has been widely applied as a biomonitoring tool, it has not been used previously in the context of bat box surveys in New Zealand. Here we present a method for collecting eDNA from bat boxes using swabs and test the efficacy of eDNA in validating bat box utilisation as confirmation of roosting. Bat boxes were deployed by the Hamilton City Council as part of the bat mitigation strategy for the Southern Links Project in southern Hamilton. The eDNA method confirmed a total of 25 boxes out of the 73 (34%) included in this study were being utilised by long-tailed bats. The results indicate that eDNA can be applied with confidence to demonstrate box use, even at very low and infrequent occupancy rates. [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]

Although new data and methodologies are gaining prominence in the field of taxonomy, the vast majority of terrestrial isopod species are still described solely based on morphology. The genus Benthana was recently revised and currently comprises 30 species found in South America. However, the broad geographic distribution of some species, coupled with the presence of few diagnostic characters, raises doubts about the validity of the described species. This study aimed to test the applicability of DNA Barcoding to assess the congruence between morphological and molecular identification of Benthana species. We analyzed 72 individuals belonging to 13 species from the scientific collection of the UFRGS Crustacean Laboratory. We obtained 24 sequences from ten Benthana species. The molecular phylogenies demonstrated that Benthana forms a paraphyletic group. Even at the species level, paraphyletic taxa were observed. Species delimitation methods revealed the presence of eight molecular entities within Benthana. Regarding genetic distances, we observed low intra-specific distances and significantly high inter-specific distances. Our results show that molecular analysis does not support the current taxonomy of Benthana. Therefore, a reassessment of the genus systematics based on integrative taxonomy is warranted. [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]

Zooplankton monitoring is important for understanding their population dynamics and life history, ecosystem health, and environmental changes. Compared with traditional morphological identification, environmental DNA (eDNA) analysis allows for more sensitive and efficient monitoring of zooplankton diversity. Previous eDNA studies have primarily used metabarcoding approaches to reveal their richness and composition, whereas its performance in predicting zooplankton abundance remains understudied. We conducted water and bulk sampling in Lake Biwa, Japan, showing that the number of sequence reads by metabarcoding moderately correlated with eDNA concentrations estimated by quantitative real-time PCR (qPCR). In addition, the eDNA read number was significantly related to cladoceran and copepod abundance estimated by microscopy sorting, although there remained too much uncertainty in the read-abundance relationship. Moreover, there was a significant difference in species composition between eDNA metabarcoding and sorting. Although our results indicated the potential applicability of eDNA metabarcoding for quantifying multiple zooplankton abundance, several methodological validations in eDNA metabarcoding would also be required to optimize its performance in the future. [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]

Invasive alien plants represent one of the five major threats to biodiversity and the disruption of ecosystems. They are introduced through various routes, starting with commercial trade. Preventing their introduction is essential to avoid the spread of new invasive plants. In this paper, we propose a new early warning DNA barcoding tool for invasive plant detection. Eight invasive alien species of European Union concern (i.e., Ludwigia grandiflora, Elodea nuttallii, Myriophyllum aquaticum, Pontederia crassipes, Ailanthus altissima, Heracleum mantegazzianum, Impatiens glandulifera, Pueraria montana) were selected and analysed. A unique DNA marker for each species was identified and amplified using species-specific primers capable of identifying the presence of alien species. To verify whether the approach could detect the presence of alien plants in urban areas from lawn clippings, mixes with typical urban spontaneous plants and invasive species were tested. In all mixes, only the invasive species was identified. This rapid detection capability will enable environmental operators to intervene promptly to contain the spread of invasive plants before they can cause significant damage to the local ecosystem. This tool could have a significant impact on the protection of local biodiversity and the integrity of urban habitats. [ABSTRACT FROM AUTHOR]

A partial mitochondrial DNA Cytochrome Oxidase subunit I (mtCOI) gene haplotype variant of the coconut rhinoceros beetle (CRB) Oryctes rhinoceros, classed as 'CRB-G (clade I)', has been the focus of much research since 2007, with reports of invasions into new Pacific Island locations (e.g., Guam, Hawaii, Solomons Islands). For numerous invasive species, inference of invasion biology via whole genome is superior to assessments via the partial mtCOI gene. Here, we explore CRB draft mitochondrial genomes (mitogenomes) from historical and recent collections, with assessment focused on individuals associated within the CRB-G (clade I) classification. We found that all Guam CRB individuals possessed the same mitogenome across all 13 protein-coding genes and differed from individuals collected elsewhere, including 'non-Guam' individuals designated as CRB-G (clade I) by partial mtCOI assessment. Two alternative ATP6 and COIII partial gene primer sets were developed to enable distinction between CRB individuals from Guam that classed within the CRB-G (clade I) haplotype grouping and CRB-G (Clade I) individuals collected elsewhere. Phylogenetic analyses based on concatenated ATP6–COIII genes showed that only Guam CRB-G (clade I) individuals clustered together, and therefore Guam was not the source of the CRB that invaded the other locations in the Pacific assessed in this study. The use of the mtCOI and/or mtCOIII genes for initial molecular diagnosis of CRB remained crucial, and assessment of more native CRB populations will further advance our ability to identify the provenance of CRB invasions being reported within the Pacific and elsewhere. [ABSTRACT FROM AUTHOR]

Nanopore raw read accuracy has improved to over 99%, making it a potential tool for metabarcoding. For broad adoption, guidelines on quality filtering are needed to ensure reliable taxonomic unit recovery. This study aims to provide those guidelines for a fungal metabarcoding context and to apply them to a case study of ectomycorrhizae in the decaying bark of Fagus sylvatica. We introduce the eNano pipeline to test two standard metabarcoding approaches: (1) Reference-based mapping leveraging UNITE's species hypothesis system (SH approach); (2) Constructing 98% OTUs (OTU approach). Our results demonstrate that both approaches are effective with Nanopore data. When using a reference database, we recommend strict mapping criteria rather than Phred-based filtering. Leveraging the SH-system further enhances reproducibility and facilitates cross-study communication. For the 98% OTUs, filtering reads at ≥Q25 is recommended. Our case study reveals that the decay gradient is a primary determinant of community composition and that specific mycorrhizal fungi colonize decaying bark. Complementing our metabarcoding results with root tip morphotypification, we identify Laccaria amethystina and Tomentella sublilacina as key ectomycorrhizae of saplings on decaying logs. These findings demonstrate that Nanopore sequencing can provide valuable ecological insights and support its broader use in fungal metabarcoding as read quality continues to improve. [ABSTRACT FROM AUTHOR]

Echinometra spp. are pantropical echinoids found in benthic marine habitat throughout the Caribbean, Atlantic, and Indo-West Pacific oceanic regions. Currently, morphology and molecular data are sparse for echinoids observed along the northeastern coast of Toco, Trinidad, where they are relatively common. Additionally, accurate species identity for Echinometra spp. remains dynamic at both northernmost and southernmost parts of the Caribbean Sea. Although distribution of sea urchins in the genus Echinometra have extensively been studied throughout the Atlantic and Indo-West Pacific, information on its range of distribution at the edge of the Caribbean Sea is lacking. In this study, the mitochondrial Cytochrome c Oxidase subunit I (mt COI) gene was amplified using polymerase chain reaction, then sequenced. Based on successfully obtained gene sequences for 581 base pairs, the echinoid species Echinometra lucunter and Echinometra viridis were identified for black and red color morphotypes from Trinidad (n  = 23) and Key Largo, Florida (n  = 6), respectively. Furthermore, these specimens were genetically identical to species identified in other studies for Puerto Rico, Panamá, Honduras, and Belize. Although morphological variations, such as spine and test color occur throughout Echinometra spp. molecular identification using the barcoding technique confirmed E. lucunter color morphs for the first time in Trinidad. Since the status of E. lucunter populations, specifically at the most northern and southern regions of the Caribbean Sea is dynamic, further studies using gene markers are essential in determining species distribution, in light of current trends in climate change. [ABSTRACT FROM AUTHOR]

The Asian hornet, Vespa velutina, is an invasive species that has not only expanded its range in Asia but has also invaded European countries, and it incurs significant costs on local apiculture. This phylogeographic study aims to trace the evolutionary trajectory of V. velutina and its close relatives; it aims to identify features that characterize an invasive species. The last successful invasion of Vespa velutina into France occurred in late May, 2002, and into South Korea in early October, 2002, which were estimated by fitting a logistic equation to the number of observations over time. The instantaneous rate of increase is 1.3667 for V. velutina in France and 0.2812 in South Korea, which are consistent with the interpretation of little competition in France and strong competition from local hornet species in South Korea. The invasive potential of two sister lineages can be compared by their distribution area when proper statistical adjustments are made to account for differences in sample size. V. velutina has a greater invasive potential than its sister lineage. The ancestor of V. velutina split into two lineages, one found in Indonesia/Malaysia and the other colonizing the Asian continent. The second lineage split into a sedentary clade inhabiting Pakistan and India and an invasive lineage colonizing much of Southeast Asia. This latter lineage gave rise to the subspecies V. v. nigrithorax, which invaded France, South Korea, and Japan. My software PGT version 1.5, which generates geophylogenies and computes geographic areas for individual taxa, is useful for understanding biogeography in general and invasive species in particular. I discussed the conceptual formulation of an index of invasiveness for a comparison between sister lineages. [ABSTRACT FROM AUTHOR]

Combating wildlife crimes in South Africa requires accurate identification of traded species and their products. Diagnostic morphological characteristics needed to identify species are often lost when specimens are processed and customs officials lack the expertise to identify species. As a potential solution, DNA barcoding can be used to identify morphologically indistinguishable specimens in forensic cases. However, barcoding is hindered by the reliance on comprehensive, validated DNA barcode reference databases, which are currently limited. To overcome this limitation, we constructed a barcode library of cytochrome c oxidase subunit 1 and cytochrome b sequences for threatened and protected mammals exploited in southern Africa. Additionally, we included closely related or morphologically similar species and assessed the database's ability to identify species accurately. Published southern African sequences were incorporated to estimate intraspecific and interspecific variation. Neighbor-joining trees successfully discriminated 94%–95% of the taxa. However, some widespread species exhibited high intraspecific distances (>2%), suggesting geographic sub-structuring or cryptic speciation. Lack of reliable published data prevented the unambiguous discrimination of certain species. This study highlights the efficacy of DNA barcoding in species identification, particularly for forensic applications. It also highlights the need for a taxonomic re-evaluation of certain widespread species and challenging genera. [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]

Simple Summary: This first comprehensive genomic analysis of rapidly growing non-tuberculous mycobacteria (RGM) isolates from Betta splendens has led to the re-identification of certain isolates and the discovery of a potential new species, Mycobacterium mucogenicum subsp. phocaicum sp. nov. The in silico analysis is consistent with and confirms previously reported in vitro studies on virulence and antibiotic susceptibility profiles. This study emphasizes the crucial role of whole genome sequencing in the accurate identification and classification of mycobacterial species. This study aims to genomically elucidate six isolates of rapidly growing non-tuberculous mycobacteria (RGM) derived from Siamese fighting fish (Betta splendens). These isolates had previously undergone phenotypic and biochemical characterization, antibiotic susceptibility testing, and in vivo virulence assessment. Initial DNA barcoding using the 16S rRNA sequence assigned these six isolates to five different species, namely Mycobacterium chelonae (BN1983), M. cosmeticum (BN1984 and N041), M. farcinogenes (SNSK5), M. mucogenicum (BN1956), and M. senegalense (BN1985). However, the identification relied solely on the highest percent identity of the 16S rRNA gene, raising concerns about the taxonomic ambiguity of these species. Comprehensive whole genome sequencing (WGS) and extended genomic comparisons using multilocus sequence typing (MLST), average nucleotide identity (ANI), and digital DNA–DNA hybridization (dDDH) led to the reclassification of BN1985 and SNSK5 as M. conceptionense while confirming BN1983 as M. chelonae and BN1984 and N041 as M. cosmeticum. Notably, the analysis of the BN1956 isolate revealed a potential new species that is proposed here as M. mucogenicum subsp. phocaicum sp. nov. Common genes encoding "mycobacterial" virulence proteins, such as PE and PPE family proteins, MCE, and YrbE proteins, were detected in all six isolates. Two species, namely M. chelonae and M. cosmeticum, appear to have horizontally acquired T6SS-II (clpB), catalase (katA), GroEL (groel), and capsule (rmlb) from distantly related environmental bacteria such as Klebsiella sp., Neisseria sp., Clostridium sp., and Streptococcus sp. This study provides the first draft genome sequence of RGM isolates currently circulating in B. splendens and underscores the necessity of WGS for the identification and classification of mycobacterial species. [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]

The fruit known as wax apple, scientifically named Syzygium samarangense (Blume) Merr. & L.M. Perry, is a well- liked agricultural product originating from Demak Regency in the Central Java Province. When it is difficult to distinguish between different forms of an organism, using DNA barcoding to authenticate the identity of species based on their DNA is an important way. The psbA-trnH intergenic spacer is a commonly used molecular approach to analyse the genetic characteristics of plant species. This study investigated the psbA-trnH intergenic spacer profile of wax apple cultivars from Demak regency. The investigation revealed that the psbA-trnH intergenic spacer sequences of the S. samarangense cultivars, namely Madu Thailand and Madu Deli Hijau, had lengths of 535 and 492 base pairs, respectively. Both cultivars demonstrate a greater nucleotide composition of deoxyadenylic acid (A) and deoxythymidylic acid (T) in comparison to deoxycytidilic acid (C) and deoxyguanylic (G). The genetic distance between S. samarangense ‘Madu Thailand’ and ‘Madu Deli Hijau’ indicates a very tight relationship, with a value of 0.000. The psbA-trnH intergenic spacer proved to be insufficient in differentiating the S. samarangense cultivars from Demak regency, mostly because to its low capacity to discern between the wax apple cultivars. [ABSTRACT FROM AUTHOR]

DNA barcoding is an effective identification tool for tree ferns with heteromorphic generations with morphologically simple gametophytic phase. A complete reference dataset which includes coverage of the target local flora is necessary for accurate identification. Three different Cyathea sp.pl. were investigated to test the utility of plastid DNA barcode regions rbcL with the intention of developing an identification system for native pteridophytes. In addition, the inter-specific and inter-generic variations among the Cyathea sp.pl. were also focused. BLAST analysis revealed the similar rbcL sequences related to C. nilgirensis Holttum, C. gigantea (Wallich ex Hook. f.) Holttum and C. crinita (Hook.) Copel and a sum of 143 sequences viz., Cyathea sp.pl. (121), Gymnosphaera sp.pl.(4), Sphaeropteris sp.pl. (1) and Alsophila sp.pl. (17). The sequences of 143 plants were retrieved from GenBank in FASTA format. The sequences were aligned using multiple sequence alignment tool MUSCLE. The inter-specific and inter-generic distance percentage were calculated using Maximum Likelihood, Minimum Evolution, UPGMA (Unweighted Pair Group Method with Arithmetic Mean) and Neighbor-Joining method. The analyses were constructed using MEGA X software. The rbcL gene provided sequence variation with its strong resolving power. This study demonstrates the overall effectiveness of DNA barcodes for species identification of tree fern genus Cyathea in the pteridophyte flora of Southern Western Ghats. [ABSTRACT FROM AUTHOR]

DNA barcoding is a popular approach that can aid in identifying species and has led to the discovery of many new amphibian species. In this study, we first performed DNA barcoding for two Hylidae family representatives (Hyla orientalis and Hyla savignyi) in the Anatolia region of Türkiye. Five species delineation tests (ABGD, GMYC, PTP, RESL, and statistical parsimony analysis) were applied, and four of them indicated that three mitochondrial lineages of Hylidae are present in Anatolia. Intraspecific genetic distances (K2P) ranged from 0.0014 to 0.0045, while the interspecific genetic distances ranged from 0.0753 to 0.1933. According to the tree topologies obtained from maximum likelihood (ML) within H. savignyi from Anatolia, two lineages can be differentiated, and further investigation is warranted as to whether these lineages represent distinct species. [ABSTRACT FROM AUTHOR]

Thai species of the pill millipede genus Hyleoglomeris Verhoeff, 1910 are reviewed, and an annotated catalogue is provided. Six new species from Thailand are described and illustrated based on morphological characteristics and molecular analyses: H. dracosphaera sp. n., H. nigromaculata sp. n., H. suwannakhuhensis sp. n., H. bomba sp. n., H. tongkerdae sp. n., and H. krasoon sp. n. They are morphologically similar, yet all can be separated based on their characteristic colour patterns, telopod structures and geographic distributions. Genetic differences in the DNA barcode region confirm their full species statuses, with interspecific COI p-distances ranging from 9.01% to 16.51% dissimilarity. We also propose the following new synonym: H. hongkhraiensis Likhitrakarn, Golovatch & Panha, 2015 = H. cavicola Likhitrakarn, Golovatch & Panha, 2015 syn. n. This is based on the low genetic divergence observed between these two taxa (2.34% COI p-distance). A new distribution map of and a key to all Hyleoglomeris species presently known to occur in Thailand are also provided. [ABSTRACT FROM AUTHOR]