Phylogenomics including new sequence data of phytoplankton-infecting chytrids reveals multiple independent lifestyle transitions across the phylum.

Parasitism is the most common lifestyle on Earth and has emerged many times independently across the eukaryotic tree of life. It is frequently found among chytrids (Chytridiomycota), which are early-branching unicellular fungi that feed osmotrophically via rhizoids as saprotrophs or parasites. Chytrids are abundant in most aquatic and terrestrial environments and fulfil important ecosystem functions. As parasites, they can have significant impacts on host populations. They cause global amphibian declines and influence the Earth's carbon cycle by terminating algal blooms. To date, the evolution of parasitism within the chytrid phylum remains unclear due to the low phylogenetic resolution of rRNA genes for the early diversification of fungi, and because few parasitic lineages have been cultured and genomic data for parasites is scarce. Here, we combine transcriptomics, culture-independent single-cell genomics and a phylogenomic approach to overcome these limitations. We newly sequenced 29 parasitic taxa and combined these with existing data to provide a robust backbone topology for the diversification of Chytridiomycota. Our analyses reveal multiple independent lifestyle transitions between parasitism and saprotrophy among chytrids and multiple host shifts by parasites. Based on these results and the parasitic lifestyle of other early-branching holomycotan lineages, we hypothesise that the chytrid last common ancestor was a parasite of phytoplankton.

Single-cell genomics reveals new rozellid lineages and supports their sister relationship to Microsporidia.

The phylum Rozellomycota has been proposed for a group of early-branching holomycotan lineages representing obligate parasites and hyperparasites of zoosporic fungi, oomycotes or phytoplankton. Given their predominantly intracellular lifestyle, rozellids are typically known from environmental ribosomal DNA data, except for the well-studied Rozella species. To date, the phylogenetic relationship between rozellids and microsporidians (Microsporidia) is not fully understood and most reliable hypotheses are based on phylogenomic analyses that incorporate the only publicly available rozellid genome of Rozella allomycis. Here, we provide genomic data of three new rozellid lineages obtained by single-cell sequencing from environmental samples and show with a phylogenomic approach that rozellids form a monophyletic group that is sister to microsporidians, corroborating the previously proposed phylum Rozellomycota. Whereas no mitochondrial genes coding for the respiratory Complex I could be found, we discovered a gene coding for a nucleotide phosphate transporter in one of the three draft genomes. The scattered absence of Complex I genes and scattered presence of nucleotide transporter genes across diverse microsporidian and rozellid lineages suggest that these adaptations to a parasitic lifestyle, which reduce the parasite's capability to synthesize ATP but enables it to steal ATP from its host, evolved independently in microsporidians and rozellids.

Spatial and phylogenetic structure of Alpine stonefly assemblages across seven habitats using DNA-species.

Stream ecosystems are spatially heterogeneous, with many different habitat patches distributed within a small area. The influence of this heterogeneity on the biodiversity of benthic insect communities is well documented; however, studies of the role of habitat heterogeneity in species coexistence and assembly remain limited. Here, we investigated how habitat heterogeneity influences spatial structure (beta biodiversity) and phylogenetic structure (evolutionary processes) of benthic stonefly (Plecoptera, Insecta) communities. We sampled 20 sites along two Alpine rivers, including seven habitats in four different reaches (headwaters, meandering, bar-braided floodplain, and lowland spring-fed). We identified 21 morphological species and delineated 52 DNA-species based on sequences from mitochondrial cox1 and nuclear ITS markers. Using DNA-species, we first analysed the patterns of variation in richness, diversity, and assemblage composition by quantifing the contribution of each reach and habitat to the overall DNA-species diversity using an additive partition analysis and distance-based redundancy analysis. Using gene-tree phylogenies, we assessed whether environmental filtering could lead to the co-occurrence of DNA-species using a two-step analysis to detect a phylogenetic signal. All four reaches significantly contributed to DNA-species richness, with the meandering reach having the highest contribution. Habitats had an effect on DNA-species diversity, where glide, riffle and, pool influenced the spatial structure of stonefly assemblage possibly due to the high habitat heterogeneity. Among the habitats, the pool showed significant phylogenetic clustering, suggesting high levels of evolutionary adaptation and strong habitat filtering. This assemblage structure may be caused by long-term stability of the habitat and the similar requirements for co-occurring species. Our study shows the importance of different habitats for the spatial and phylogenetic structure of stonefly assemblage and sheds light on the habitat-specific diversity that may help improve conservation practices.

Trade-offs between reducing complex terminology and producing accurate interpretations from environmental DNA: Comment on "Environmental DNA: What's behind the term?" by Pawlowski et al., (2020).

In a recent paper, "Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring," Pawlowski et al. argue that the term eDNA should be used to refer to the pool of DNA isolated from environmental samples, as opposed to only extra-organismal DNA from macro-organisms. We agree with this view. However, we are concerned that their proposed two-level terminology specifying sampling environment and targeted taxa is overly simplistic and might hinder rather than improve clear communication about environmental DNA and its use in biomonitoring. This terminology is based on categories that are often difficult to assign and uninformative, and it overlooks a fundamental distinction within eDNA: the type of DNA (organismal or extra-organismal) from which ecological interpretations are derived.

Temporal dynamics of freshwater planktonic parasites inferred using a DNA metabarcoding time-series.

Parasites are important components of biodiversity and contributors to ecosystem functioning, but are often neglected in ecological studies. Most studies examine model parasite systems or single taxa, thus our understanding of community composition is lacking. Here, the seasonal and annual dynamics of parasites was quantified using a 5-year metabarcoding time-series of freshwater plankton, collected weekly. We first identified parasites in the dataset using literature searches of the taxonomic match and using sequence metadata from the National Center for Biotechnology Information (NCBI) nucleotide database. In total, 441 amplicon sequence variants (belonging to 18 phyla/clades) were classified as parasites. The four phyla/clades with the highest relative read abundance and richness were Chytridiomycota, Dinoflagellata, Oomycota and Perkinsozoa. Relative read abundance of total parasite taxa, Dinoflagellata and Perkinsozoa significantly varied with season and was highest in summer. Parasite richness varied significantly with season and year, and was generally lowest in spring. Each season had distinct parasite communities, and the difference between summer and winter communities was most pronounced. Combining DNA metabarcoding with searches of the literature and NCBI metadata allowed us to characterize parasite diversity and community dynamics and revealed the extent to which parasites contribute to the diversity of freshwater plankton communities.

Encapsulation of Anguillicola crassus reduces the abundance of adult parasite stages in the European eel (Anguilla anguilla).

Encapsulation of the parasitic nematode Anguillicola crassus Kuwahara, Niimi & Hagaki is commonly observed in its native host, the Japanese eel (Anguilla japonica Temminck & Schlegel). Encapsulation has also been described in a novel host, the European eel (A. anguilla L.), and there is evidence that encapsulation frequency has increased since the introduction of A. crassus. We examined whether encapsulation of A. crassus provides an advantage to its novel host in Lake Müggelsee, NE Germany. We provide the first evidence that encapsulation was associated with reduced abundance of adult A. crassus. This pattern was consistent in samples taken 3 months apart. There was no influence of infection on the expression of the two metabolic genes studied, but the number of capsules was negatively correlated with the expression of two mhc II genes of the adaptive immune response, suggesting a reduced activation. Interestingly, eels that encapsulated A. crassus had higher abundances of two native parasites compared with non-encapsulating eels. We propose that the response of A. anguilla to infection by A. crassus may interfere with its reaction to other co-occurring parasites.

Revisiting the phylogenetic position of Caullerya mesnili (Ichthyosporea), a common Daphnia parasite, based on 22 protein-coding genes.

Caullerya mesnili is a common and virulent parasite of the water flea, Daphnia. It was classified within the Haplosporidia (Rhizaria) for over a century. However, a recent molecular phylogeny based on the 18S rRNA gene suggested it belonged to the Ichthyosporea, a class of protists closely related to animals within the Opisthokonta clade. The exact phylogenetic position of C. mesnili remained uncertain because it appeared in the 18S rRNA tree with a very long branch and separated from all other taxa, suggesting that its position could be artifactual. A better understanding of its phylogenetic position has been constrained by a lack of molecular markers and the difficulty of obtaining a suitable quantity and quality of DNA from in vitro cultures, as this intracellular parasite cannot be cultured without its host. We isolated and collected spores of C. mesnili and sequenced genomic libraries. Phylogenetic analyses of a newly generated multi-protein data set (22 proteins, 4998 amino acids) and of sequences from the 18S rRNA gene both placed C. mesnili within the Ichthyophonida sub-clade of Ichthyosporea, as sister-taxon to Abeoforma whisleri and Pirum gemmata. Our study highlights the utility of metagenomic approaches for obtaining genomic information from intracellular parasites and for more accurate phylogenetic placement in evolutionary studies.

Daphnia galeata responds to the exposure to an ichthyosporean gut parasite by down-regulation of immunity and lipid metabolism.

BACKGROUND: Regulatory circuits of infection in the emerging experimental model system, water flea Daphnia and their microparasites, remain largely unknown. Here we provide the first molecular insights into the response of Daphnia galeata to its highly virulent and common parasite Caullerya mesnili, an ichthyosporean that infects the gut epithelium. We generated a transcriptomic dataset using RNAseq from parasite-exposed (vs. control) Daphnia, at two time points (4 and 48 h) after parasite exposure. RESULTS: We found a down-regulation of metabolism and immunity-related genes, at 48 h (but not 4 h) after parasite exposure. These genes are involved in lipid metabolism and fatty acid biosynthesis, as well as microbe recognition (e.g. c-type lectins) and pathogen attack (e.g. gut chitin). CONCLUSIONS: General metabolic suppression implies host energy shift from reproduction to survival, which is in agreement with the known drastic reduction in Daphnia fecundity after Caullerya infection. The down-regulation of gut chitin indicates a possible interaction between the peritrophic matrix and the evading host immune system. Our study provides the first description of host transcriptional responses in this very promising host-parasite experimental system.

Colonization and diversification of aquatic insects on three Macaronesian archipelagos using 59 nuclear loci derived from a draft genome.

The study of processes driving diversification requires a fully sampled and well resolved phylogeny, although a lack of phylogenetic markers remains a limitation for many non-model groups. Multilocus approaches to the study of recent diversification provide a powerful means to study the evolutionary process, but their application remains restricted because multiple unlinked loci with suitable variation for phylogenetic or coalescent analysis are not available for most non-model taxa. Here we identify novel, putative single-copy nuclear DNA (nDNA) phylogenetic markers to study the colonization and diversification of an aquatic insect species complex, Cloeon dipterum L. 1761 (Ephemeroptera: Baetidae), in Macaronesia. Whole-genome sequencing data from one member of the species complex were used to identify 59 nDNA loci (32,213 base pairs), followed by Sanger sequencing of 29 individuals sampled from 13 islands of three Macaronesian archipelagos. Multispecies coalescent analyses established six putative species. Three island species formed a monophyletic clade, with one species occurring on the Azores, Europe and North America. Ancestral state reconstruction indicated at least two colonization events from the mainland (to the Canaries, respectively Azores) and one within the archipelago (between Madeira and the Canaries). Random subsets of the 59 loci showed a positive linear relationship between number of loci and node support. In contrast, node support in the multispecies coalescent tree was negatively correlated with mean number of phylogenetically informative sites per locus, suggesting a complex relationship between tree resolution and marker variability. Our approach highlights the value of combining genomics, coalescent-based phylogeography, species delimitation, and phylogenetic reconstruction to resolve recent diversification events in an archipelago species complex.

City-Dwellers and Country Folks: Lack of Population Differentiation Along an Urban-Rural Gradient in the Mosquito Culex pipiens (Diptera: Culicidae).

Mosquitoes (Diptera, Culicidae) occur in natural, urban, and peri-urban areas throughout the globe. Although the characteristics of urban and peri-urban habitats differ from those of natural habitats in many ways (e.g., fragmentation, pollution, noise, and light), few studies have examined the population connectivity of mosquitoes in urban areas. To obtain an overview of the species composition, we sampled mosquitoes from 23 sites in and around the city of Berlin, Germany. Of 23 species, five occurred in urban, 10 in peri-urban, and 20 in rural areas. Culex pipiens Linnaeus (Diptera: Culicidae) was the most common species collected (75% of all individuals) and occurred in all habitats. Hence this species was selected to be analysed at 10 microsatellite markers. There was no significant differentiation (FST = 0.016, P = 0.9) or isolation by distance (P = 0.06) among Cx. pipiens populations along an urban-rural gradient. The only significant differences detected were between Cx. pipiens and a laboratory population of Cx. pipiens f. molestus (pairwise FST = 0.114-0.148, P ≤ 0.001 in all comparisons), suggesting that the markers chosen were suitable for the identification of population differentiation. Our results indicate that Cx. pipiens gene flow is widespread within and among urban, peri-urban, and rural areas and that urban habitat does not necessarily impede or enhance gene flow among these populations.

Molecular phylogeny and timing of diversification in Alpine Rhithrogena (Ephemeroptera: Heptageniidae).

BACKGROUND: Larvae of the Holarctic mayfly genus Rhithrogena Eaton, 1881 (Ephemeroptera, Heptageniidae) are a diverse and abundant member of stream and river communities and are routinely used as bio-indicators of water quality. Rhithrogena is well diversified in the European Alps, with a number of locally endemic species, and several cryptic species have been recently detected. While several informal species groups are morphologically well defined, a lack of reliable characters for species identification considerably hampers their study. Their relationships, origin, timing of speciation and mechanisms promoting their diversification in the Alps are unknown. RESULTS: Here we present a species-level phylogeny of Rhithrogena in Europe using two mitochondrial and three nuclear gene regions. To improve sampling in a genus with many cryptic species, individuals were selected for analysis according to a recent DNA-based taxonomy rather than traditional nomenclature. A coalescent-based species tree and a reconstruction based on a supermatrix approach supported five of the species groups as monophyletic. A molecular clock, mapped on the most resolved phylogeny and calibrated using published mitochondrial evolution rates for insects, suggested an origin of Alpine Rhithrogena in the Oligocene/Miocene boundary. A diversification analysis that included simulation of missing species indicated a constant speciation rate over time, rather than any pronounced periods of rapid speciation. Ancestral state reconstructions provided evidence for downstream diversification in at least two species groups. CONCLUSIONS: Our species-level analyses of five gene regions provide clearer definitions of species groups within European Rhithrogena. A constant speciation rate over time suggests that the paleoclimatic fluctuations, including the Pleistocene glaciations, did not significantly influence the tempo of diversification of Alpine species. A downstream diversification trend in the hybrida and alpestris species groups supports a previously proposed headwater origin hypothesis for aquatic insects.

Sex-specific gene expression in the mosquito Culex pipiens f. molestus in response to artificial light at night.

BACKGROUND: Artificial light at night (ALAN) is a typical feature of urban areas and most organisms living in urban or suburban habitats are exposed to low levels of ALAN. Light is one of the most important environmental cues that organisms use to regulate their activities. Studies have begun to quantify the influence of ALAN on the behavior and ecology of organisms, but research on the effects at the molecular level remains limited. Mosquitoes in the Culex pipiens complex (Diptera, Culicidae) are widespread and abundant in urban areas where they are potential disease vectors. It is thus of particular interest to understand how ALAN may influence biologically and ecologically relevant traits. RESULTS: We used RNAseq to evaluate the transcriptome response in a Cx. pipiens f. molestus laboratory population that was exposed to near-natural light conditions (light:dark L16:D8 hours, "control") and ALAN conditions with 3 h of constant low-level light at night (L16 + Llow3:D5 hours, "low-light"). The resulting transcripts were mapped to the reference genome of the closely related Culex quinquefasciatus. Female expression patterns differed significantly between control and treatment conditions at five genes although none showed an absolute fold change greater than two (FC > 2). In contrast, male expression differed at 230 genes (74 with FC > 2). Of these, 216 genes (72 with FC > 2) showed reduced expression in the low-light treatment, most of which were related to gametogenesis, lipid metabolism, and immunity. Of the 14 genes (two with FC > 2) with increased expression, only five had any functional annotation. There was a pronounced sex-bias in gene expression regardless of treatment, with 11,660 genes (51 % of annotated genes; 8694 with FC > 2; 48 % of annotated genes) differentially expressed between males and females, including 14 genes of the circadian clock. CONCLUSION: Our data suggest a stronger response to artificial light by males of Cx. pipiens f. molestus than by females, and that a wide range of physiological pathways may be affected by ALAN at the molecular level. The fact that differences in gene expression appear to be sex-specific may have a strong influence at the population level.

Freshwater biodiversity and aquatic insect diversification.

Inland waters cover less than 1% of Earth's surface but harbor more than 6% of all insect species: Nearly 100,000 species from 12 orders spend one or more life stages in freshwater. Little is known about how this remarkable diversity arose, although allopatric speciation and ecological adaptation are thought to be primary mechanisms. Freshwater habitats are highly susceptible to environmental change and exhibit marked ecological gradients. Standing waters appear to harbor more dispersive species than running waters, but there is little understanding of how this fundamental ecological difference has affected diversification. In contrast to the lack of evolutionary studies, the ecology and habitat preferences of aquatic insects have been intensively studied, in part because of their widespread use as bioindicators. The combination of phylogenetics with the extensive ecological data provides a promising avenue for future research, making aquatic insects highly suitable models for the study of ecological diversification.

The haplotype-resolved Prymnesium parvum (type B) microalga genome reveals the genetic basis of its fish-killing toxins.

The catastrophic loss of aquatic life in the Central European Oder River in 2022, caused by a toxic bloom of the haptophyte microalga Prymnesium parvum (in a wide sense, s.l.), underscores the need to improve our understanding of the genomic basis of the toxin. Previous morphological, phylogenetic, and genomic studies have revealed cryptic diversity within P. parvum s.l. and uncovered three clade-specific (types A, B, and C) prymnesin toxins. Here, we used state-of-the-art long-read sequencing and assembled the first haplotype-resolved diploid genome of a P. parvum type B from the strain responsible for the Oder disaster. Comparative analyses with type A genomes uncovered a genome-size expansion driven by repetitive elements in type B. We also found conserved synteny but divergent evolution in several polyketide synthase (PKS) genes, which are known to underlie toxin production in combination with environmental cues. We identified an approximately 20-kbp deletion in the largest PKS gene of type B that we link to differences in the chemical structure of types A and B prymnesins. Flow cytometry and electron microscopy analyses confirmed diploidy in the Oder River strain and revealed differences to closely related strains in both ploidy and morphology. Our results provide unprecedented resolution of strain diversity in P. parvum s.l. and a better understanding of the genomic basis of toxin variability in haptophytes. The reference-quality genome will enable us to better understand changes in microbial diversity in the face of increasing environmental pressures and provides a basis for strain-level monitoring of invasive Prymnesium in the future.

Environmental DNA, hydrochemistry and stable water isotopes as integrative tracers of urban ecohydrology.

Urbanization and the persistent environmental changes present a major challenge for urban freshwaters and availability of water for humans and wildlife. In order to increase understanding of urban ecohydrology, we investigated the variability of planktonic bacteria and benthic diatoms - as two key biological indicators - coupled with insights from hydrochemistry and stable water isotopes across four urban streams characterized by different dominant water sources in Berlin, the German capital, over a period of one year (2021-2022). DNA metabarcoding results show that substantial spatio-temporal variability exists across urban streams in terms of microbial diversity and richness, with clear links to abiotic factors and nutrient concentrations. Bacterial communities showed clear distinction between effluent-impacted and non-effluent impacted streams as well as clear seasonal turnover. In-stream benthic diatom assemblages also showed robust seasonal variation as well as high species diversity. Our multiple-tracer approach is relevant for emerging questions regarding the increased use of treated effluent to supplement declining baseflows, the assessment of stream restoration projects and the impact of storm drainage and surface pollution on aquatic ecosystem health. eDNA analysis allows analysis of spatial and temporal patterns not feasibly studied with traditional analyses of macroinvertebrates. This can ultimately be leveraged for future water resource management and restoration planning and monitoring of urban freshwater systems across metropolitan areas.

Unpredicted ecosystem response to compound human impacts in a European river.

Climate change elevates the threat of compound heat and drought events, with their ecological and socioeconomic impacts exacerbated by human ecosystem alterations such as eutrophication, salinization, and river engineering. Here, we study how multiple stressors produced an environmental disaster in a large European river, the Oder River, where a toxic bloom of the brackish-water planktonic haptophyte Prymnesium parvum (the "golden algae") killed approximately 1000 metric tons of fish and most mussels and snails. We uncovered the complexity of this event using hydroclimatic data, remote sensing, cell counts, hydrochemical and toxin analyses, and genetics. After incubation in impounded upstream channels with drastically elevated concentrations of salts and nutrients, only a critical combination of chronic salt and nutrient pollution, acute high water temperatures, and low river discharge during a heatwave enabled the riverine mass proliferation of B-type P. parvum along a 500 km river section. The dramatic losses of large filter feeders and the spreading of vegetative cells and resting stages make the system more susceptible to new harmful algal blooms. Our findings show that global warming, water use intensification, and chronic ecosystem pollution could increase likelihood and severity of such compound ecoclimatic events, necessitating consideration in future impact models.

Effects of predation pressure and resource use on morphological divergence in omnivorous prey fish.

BACKGROUND: Body shape is one of the most variable traits of organisms and responds to a broad array of local selective forces. In freshwater fish, divergent body shapes within single species have been repeatedly observed along the littoral-pelagic axes of lakes, where the structural complexity of near shore habitats provides a more diverse set of resources compared to the open-water zones. It remains poorly understood whether similar resource-driven polymorphism occurs among lakes that vary in structural complexity and predation pressure, and whether this variation is heritable. Here, we analyzed body shape in four populations of omnivorous roach (Rutilus rutilus) inhabiting shallow lakes. We tested the relationship between body shape, gradients of resources, predation pressure, and, in a subset of two lakes, diet composition. We used genome scans of 331 polymorphic AFLP markers to test whether there was a heritable component to the observed morphological diversification. RESULTS: Body shape differed among lakes and was significantly correlated to differences in predation pressure. Roach from the lake with highest predation pressure were most divergent from the average body shape of all populations, characterized by a more streamlined body and caudally inserted dorsal fins; features that facilitate predator escape. Surprisingly, diet composition was not associated with morphology. AFLP analysis revealed weak genetic differentiation among lakes and no isolation by distance (IBD). Outlier analysis detected three loci under positive selection with differing frequencies in the four populations. General linear models did not support an association of lake-specific genotypes with morphological variation. CONCLUSION: Body shape was divergent among lakes, suggesting that processes previously reported from within single lakes may also be operating at the scale of whole lakes. We found no evidence for body shape being heritable, although sample size was small in these natural populations. Rather than habitat structure and diet, we conclude that predation had a stronger effect on the prevalence of local morphotypes. A variable morphotype facilitating the efficient uptake of a variety of spatially and temporarily scattered resources seems to be favored in these small aquatic systems.

Endemism and diversification in freshwater insects of Madagascar revealed by coalescent and phylogenetic analysis of museum and field collections.

The biodiversity and endemism of Madagascar are among the most extraordinary and endangered in the world. This includes the island's freshwater biodiversity, although detailed knowledge of the diversity, endemism, and biogeographic origin of freshwater invertebrates is lacking. The aquatic immature stages of mayflies (Ephemeroptera) are widely used as bio-indicators and form an important component of Malagasy freshwater biodiversity. Many species are thought to be microendemics, restricted to single river basins in forested areas, making them particularly sensitive to habitat reduction and degradation. The Heptageniidae are a globally diverse family of mayflies (>500 species) but remain practically unknown in Madagascar except for two species described in 1996. The standard approach to understanding their diversity, endemism, and origin would require extensive field sampling on several continents and years of taxonomic work followed by phylogenetic analysis. Here we circumvent this using museum collections and freshly collected individuals in a combined approach of DNA taxonomy and phylogeny. The coalescent-based GMYC analysis of DNA barcode data (mitochondrial COI) revealed 14 putative species on Madagascar, 70% of which were microendemics. A phylogenetic analysis that included African and Asian species and data from two mitochondrial and four nuclear loci indicated the Malagasy Heptageniidae are monophyletic and sister to African species. The genus Compsoneuria is shown to be paraphyletic and the genus Notonurus is reinstalled for African and Malagasy species previously placed in Compsoneuria. A molecular clock excluded a Gondwanan vicariance origin and instead favoured a more recent overseas colonization of Madagascar. The observed monophyly and high microendemism highlight their conservation importance and suggest the DNA-based approach can rapidly provide information on the diversity, endemism, and origin of freshwater biodiversity. Our results underline the important role that museum collections can play in molecular studies, especially in critically endangered biodiversity hotspots like Madagascar where entire species or populations may go extinct very quickly.

The effect of geographical scale of sampling on DNA barcoding.

Eight years after DNA barcoding was formally proposed on a large scale, CO1 sequences are rapidly accumulating from around the world. While studies to date have mostly targeted local or regional species assemblages, the recent launch of the global iBOL project (International Barcode of Life), highlights the need to understand the effects of geographical scale on Barcoding's goals. Sampling has been central in the debate on DNA Barcoding, but the effect of the geographical scale of sampling has not yet been thoroughly and explicitly tested with empirical data. Here, we present a CO1 data set of aquatic predaceous diving beetles of the tribe Agabini, sampled throughout Europe, and use it to investigate how the geographic scale of sampling affects 1) the estimated intraspecific variation of species, 2) the genetic distance to the most closely related heterospecific, 3) the ratio of intraspecific and interspecific variation, 4) the frequency of taxonomically recognized species found to be monophyletic, and 5) query identification performance based on 6 different species assignment methods. Intraspecific variation was significantly correlated with the geographical scale of sampling (R-square = 0.7), and more than half of the species with 10 or more sampled individuals (N = 29) showed higher intraspecific variation than 1% sequence divergence. In contrast, the distance to the closest heterospecific showed a significant decrease with increasing geographical scale of sampling. The average genetic distance dropped from > 7% for samples within 1 km, to < 3.5% for samples up to > 6000 km apart. Over a third of the species were not monophyletic, and the proportion increased through locally, nationally, regionally, and continentally restricted subsets of the data. The success of identifying queries decreased with increasing spatial scale of sampling; liberal methods declined from 100% to around 90%, whereas strict methods dropped to below 50% at continental scales. The proportion of query identifications considered uncertain (more than one species < 1% distance from query) escalated from zero at local, to 50% at continental scale. Finally, by resampling the most widely sampled species we show that even if samples are collected to maximize the geographical coverage, up to 70 individuals are required to sample 95% of intraspecific variation. The results show that the geographical scale of sampling has a critical impact on the global application of DNA barcoding. Scale-effects result from the relative importance of different processes determining the composition of regional species assemblages (dispersal and ecological assembly) and global clades (demography, speciation, and extinction). The incorporation of geographical information, where available, will be required to obtain identification rates at global scales equivalent to those in regional barcoding studies. Our result hence provides an impetus for both smarter barcoding tools and sprouting national barcoding initiatives-smaller geographical scales deliver higher accuracy.