COI Metabarcoding Research Articles

Long-distance Southern Ocean environmental DNA (eDNA) transect provides insights into spatial marine biota and invasion pathways for non-native species

The Southern Ocean surrounding Antarctica harbours some of the most pristine marine environments remaining, but is increasingly vulnerable to anthropogenic pressures, climate change, and invasion by non-native species. Monitoring biotic responses to cumulative impacts requires temporal and spatial baselines and ongoing monitoring - traditionally, this has been obtained by continuous plankton recorder (CPR) surveys. Here, we conduct one of the longest environmental DNA (eDNA) transects yet, spanning over 3000 nautical miles from Hobart (Australia) to Davis Station (Antarctica). We evaluate eDNA sampling strategies for long-term open ocean biomonitoring by comparing two water volume and filter pore size combinations: large (12 l with 20 μm) and small (2 l with 0.45 μm). Employing a broad COI metabarcoding assay, we found the large sample/pore combination was better suited to open ocean monitoring, detecting more target DNA and rare or low abundance species. Comparisons with four simultaneously conducted CPR transects revealed that eDNA detections were more diverse than CPR, with 7 (4 unique) and 4 (1 unique) phyla detections respectively. While both methods effectively delineated biodiversity patterns across the Southern Ocean, eDNA enables surveys in the presence of sea-ice where CPR cannot be conducted. Accordingly, 16 species of concern were detected along the transect using eDNA, notably in the Antarctic region (south of 60°S). These were largely attributed to hull biofouling, a recognized pathway for marine introductions into Antarctica. Given the vulnerability of Antarctic environments to potential introductions in a warming Southern Ocean, this work underscores the importance of continued biosecurity vigilance. We advocate integrating eDNA metabarcoding with long-term CPR surveys in the Southern Ocean, emphasising the urgency of its implementation. We anticipate temporal and spatial interweaving of CPR, eDNA, and biophysical data will generate a more nuanced picture of Southern Ocean ecosystems, with significant implications for the conservation and preservation of Antarctic ecosystems.

Mining exploration infrastructure affects biophysical habitat characteristics and ground-dwelling arthropod communities

Monitoring of environmental impacts of mining activities typically focuses on the main operation footprint, neglecting exploration infrastructure like tracks, roads, and drill pads. These areas are cleared of native vegetation and impacts on the surrounding environment can be both cumulative and enigmatic. Here, we study the impacts of mining exploration infrastructure on habitat characteristics and ground-dwelling arthropod communities in the Midwest region of Western Australia. The study was conducted at three mine sites, each with three infrastructure types: maintained tracks, unmaintained tracks, and drill pads along transects extending 100 m away from the disturbance into remnant vegetation. Habitat characteristics were measured, and arthropods collected from pitfall traps along these transects and identified using COI metabarcoding. The overall arthropod community and two indicator groups, ants (Formicidae) and springtails (Collembola) - were used to measure arthropod responses to changes in response to habitat disturbance. Whilst changes in habitat were only visible to 10 m from the disturbance, impacts on arthropod communities could be detected up to 100 m into the remnant vegetation, and these responses were more complex. In general, we found similar patterns expressed in the compositional changes for arthropods overall and between our chosen indicator groups, but they were not the same across all sites and infrastructure types. Our results demonstrate the utility of bulk arthropod metabarcoding and different arthropod indicator groups for documenting the effects of fine-scale habitat destruction, degradation, or disturbance. They also highlight the need to monitor the negative impacts of mineral exploration on the environment.

Life goes on: Spatial heterogeneity promotes biodiversity in an urbanized coastal marine ecosystem.

Both human populations and marine biodiversity are concentrated along coastlines, with growing conservation interest in how these ecosystems can survive intense anthropogenic impacts. Tropical urban centres provide valuable research opportunities because these megacities are often adjacent to mega-diverse coral reef systems. The Pearl River Delta is a prime exemplar, as it encompasses one of the most densely populated and impacted regions in the world and is located just northwest of the Coral Triangle. However, the spatial and taxonomic complexity of this biodiversity, most of which is small, cryptic in habitat and poorly known, make comparative analyses challenging. We deployed standardized settlement structures at seven sites differing in the intensity of human impacts and used COI metabarcoding to characterize benthic biodiversity, with a focus on metazoans. We found a total of 7184 OTUs, with an average of 665 OTUs per sampling unit; these numbers exceed those observed in many previous studies using comparable methods, despite the location of our study in an urbanized environment. Beta diversity was also high, with 52% of the OTUs found at just one site. As expected, we found that the sites close to point sources of pollution had substantially lower diversity (44% less) relative to sites bathed in less polluted oceanic waters. However, the polluted sites contributed substantially to the total animal diversity of the region, with 25% of all OTUs occurring only within polluted sites. Further analysis of Arthropoda, Annelida and Mollusca showed that phylogenetic clustering within a site was common, suggesting that environmental filtering reduced biodiversity to a subset of lineages present within the region, a pattern that was most pronounced in polluted sites and for the Arthropoda. The water quality gradients surrounding the PRD highlight the unique role of insitu studies for understanding the impacts of complex urbanization pressures on biodiversity.

Comparison of DNA metabarcoding and morphological diet analysis of lumpfish (Cyclopterus lumpus) as methods of estimating sea lice cleaning efficacy inside commercial Atlantic salmon (Salmo salar) sea cages

Lumpfish (Cyclopterus lumpus) are often used as cleaner fish inside salmonid sea cages for biological control of ectoparasitic sea lice. We compared traditional morphological diet assessments with DNA metabarcoding as methods of estimating lumpfish cleaning efficacy as a function of its size, and of its ingestion of alternative prey. Traditional morphological analysis was performed on 122 ethanol-preserved stomachs. The stomachs were removed in the field - following eDNA protocols – from lumpfish collected from industrial Atlantic salmon sea cages in southern Newfoundland. Morphological stomach analysis gave semi-quantitative estimates of prey counts and/or biomass at the order or family level; it showed that shorter lumpfish ate more gammarid amphipods whereas longer lumpfish ate more formulated feed pellets designed for salmon. No sea lice were confidentially morphologically identified in the lumpfish stomachs. In contrast, COI metabarcoding was able to confidently identify the presence of the salmon louse (Lepeophtheirus salmonis) in 61 of 82 successfully barcoded lumpfish stomachs. Metabarcoding showed that diurnally-migrating krill and gammarid amphipods were present in more stomachs in 2018 whereas formulated feed pellets – identified from COI sequences classified as domestic poultry and non-indigenous fish species - were present in more stomachs in 2020. We argue that, when weighed by the total number of reads, the counts per stomach of the number of Global Estimated Sequence Variants (ESV) classified as a particular prey species could be used to estimate the single-species indices of abundance needed for lumpfish breeding values. The number of ESVs classified as the salmon louse could be used to estimate an index of lumpfish cleaning efficacy. As proof-of-concept, we show that 83% of the variance in the number of Norwegian krill heads observed per stomach was explained by two weighed covariates: 1) Norwegian krill ESV counts and 2) lumpfish stomach contents weight.

DNA metabarcoding of light trap samples vs. morphological species identification

Abstract DNA metabarcoding has developed into a commonly used tool for biodiversity assessment and monitoring. How results from DNA metabarcoding are compared with studies based on ‘classic’, in most cases morphological species identification, is still unclear. Studies investigating species detection against a known baseline are virtually non‐existent. In this study, we used light trap samples collected in eastern Austria to investigate the concordance between morphological species lists and results obtained from COI metabarcoding using the Illumina MiSeq platform. Two primer combinations of different lengths (313 and 205 bp) were compared to assess the influence of amplicon length. Species detection rates ranged between 0.38 and 0.69; the shorter amplicon had on average higher species detection rates compared with the longer amplicon. Singleton species were less likely to be detected through metabarcoding. The major determinant for a species to be detected was its biomass, viz. smaller species had a lower chance to be detected. However, there is also evidence of taxonomic bias on the level of superfamilies. While the influence of biomass is to be expected, the presence of taxonomic bias gives reason for concern and requires further studies. Such a bias can be of significance when metabarcoding is used to determine conservation measures. Ordination analyses of all sampling sites showed that as far as community ecology is concerned, the overall pattern obtained from the full species list was mostly preserved in our metabarcoding results.

Amazonian ichthyoplankton assessment via DNA metabarcoding: A baseline for detecting spawning sites of migratory fishes

The Neotropical fish fauna comprises approximately 6080 species, of which almost 3000 inhabit the Amazon River basin. However, the construction of hydroelectric dams has threatened this biodiversity by habitat fragmentation. To uncover the putative effect of river impoundment on migratory fish species, over 5400 eggs and larvae were collected from the Teles Pires River, including five tributaries and the hydroelectric reservoir, which were identified using the COI metabarcoding approach. Identification of early life stages allowed the detection of >40 fish species, including at least 27 migratory species. Fish eggs were assigned to nine families, 13 genera, and 23 species, whereas larvae were attributed to 12 families, 18 genera, and 34 species. Eggs and larvae of migratory fishes were primarily present in tributaries upstream of the dam, with fewer in the reservoir. A putative impact of the dam on the ichthyoplankton community was detected, with a significant difference found between the reservoir and tributaries for larval species richness (r = 0.822, p = 0.0211). In contrast, no significant difference was recovered among tributaries for both eggs (r = 0.213, p = 0.2657) and larvae (r = 0.378, p = 0.0696). However, a change in ichthyoplankton assemblage was observed between tributaries, as revealed by beta diversity analyses. These results highlight the potential of ichthyoplankton metabarcoding for studying and biomonitoring of migratory fishes, as well as providing relevant data concerning the identification of spawning sites to set guidelines for fisheries management and effective conservation of fish populations.

Contaminants reach everywhere: Fish dietary samples should be surface decontaminated prior to molecular diet analysis.

Knowledge of trophic interaction is necessary to understand the dynamics of ecosystems and develop ecosystem-based management. The key data to measure these interactions should come from large-scale diet analyses with good taxonomic resolution. To that end, molecular methods that analyze prey DNA from guts and feces provide high-resolution dietary taxonomic data. However, molecular diet analysis may also produce unreliable results if the samples are contaminated by external sources of DNA. Employing the freshwater European whitefish (Coregonus lavaretus) as a tracer for sample contamination, we studied the possible route of whitefish in beaked redfish (Sebastes mentella) guts sampled in the Barents Sea. We used whitefish-specific COI primers for diagnostic analysis, and fish-specific 12S and metazoa-specific COI primers for metabarcoding analyses of intestine and stomach contents of fish samples that were either not cleaned, water cleaned, or bleach cleaned after being in contact with whitefish. Both the diagnostic and COI metabarcoding revealed clear positive effects of cleaning samples as whitefish were detected in significantly higher numbers of uncleaned samples compared to water or bleach-cleaned samples. Stomachs were more susceptible to contamination than intestines and bleach cleaning reduced the frequency of whitefish contamination. Also, the metabarcoding approach detected significantly more reads of whitefish in the stomach than in intestine samples. The diagnostic analysis and COI metabarcoding detected contaminants in a higher and comparable number of gut samples than the 12S-based approach. Our study underlines thus the importance of surface decontamination of aquatic samples to obtain reliable diet information from molecular data.

Metabarcoding the eukaryotic community of a threatened, iconic Mediterranean habitat: Posidonia oceanica seagrass meadows

Against the accelerating pace of worldwide species extinction, reliable biodiversity assessments are critical, both as baselines and to track potential declines. DNA metabarcoding techniques allow for fast and comprehensive assessment of biodiversity in both terrestrial and marine habitats. However, these methods need to be adapted and standardised for each ecosystem in order to be effective. Seagrass meadows are among the most diverse marine habitats and are irreplaceable in terms of the ecosystem services they provide, yet metabarcoding has never been implemented for these systems. In this study, we developed and tested a protocol for metabarcoding the eukaryotic community of meadows of the iconic species, Posidonia oceanica L. (Delile). This seagrass is the main habitat-forming species in Mediterranean coastal waters and is known for its high diversity due to the structural complexity of its canopy and rhizome structures. This habitat is experiencing a range-wide retreat, and there is an urgent need for fast and efficient methods for its biomonitoring and detection of early changes. Our proposed method involves direct sampling of the community, collecting and processing the leaves and rhizome strata separately. To test the utility of the method in distinguishing between different meadow conditions, we sampled two distinct meadows that differ in their prevailing wind and surge conditions, and a nearby rocky reef for comparison. We then adapted a method and pipeline for COI metabarcoding using generalist primers that target the eukaryote diversity present. We detected a high diversity in the two meadows analysed (3,350 molecular operational taxonomic units, dominated by Metazoa and Archaeplastida) and a clear differentiation of the seagrass samples from those of the nearby rocky reefs. The leaves and rhizomes harboured clearly distinct assemblages, and differences were also detected between the two meadows sampled. This new tool has the potential to deliver big biodiversity data for seagrass habitats in a fast and efficient way, which is crucial for the implementation of protection and management measures for this key coastal habitat.

High taxonomic diversity and miniaturization in benthic communities under persistent natural CO2 disturbances.

Metabarcoding techniques have revolutionized ecological research in recent years, facilitating the differentiation of cryptic species and revealing previously hidden diversity. In the current scenario of climate change and ocean acidification, biodiversity loss is one of the main threats to marine ecosystems. Here, we explored the effects of ocean acidification on marine benthic communities using DNA metabarcoding to assess the diversity of algae and metazoans. Specifically, we examined the natural pH gradient generated by the Fuencaliente CO2 vent system, located near La Palma Island (Canary Islands). High-resolution COI metabarcoding analyses revealed high levels of taxonomic diversity in an acidified natural area for the first time. This high number of species arises from the detection of small and cryptic species that were previously undetectable by other techniques. Such species are apparently tolerant to the acidification levels expected in future oceans. Hence and following our results, future subtropical communities are expected to keep high biodiversity values under an acidification scenario, although they will tend toward overall miniaturization due to the dominance of small algal and invertebrate species, leading to changes in ecosystem functions.

Specific and Intraspecific Diversity of Symphypleona and Neelipleona (Hexapoda: Collembola) in Southern High Appalachia (USA)

Collembola, commonly known as springtails, are important detritivores, abundant in leaf litter and soil globally. Springtails are wingless hexapods with many North American species having wide distributions ranging from as far as Alaska to Mexico. Here, we analyze the occurrence and intraspecific diversity of springtails with a globular body shape (Symphypleona and Neelipleona), in southern high Appalachia, a significant biodiversity hotspot. The peaks of high Appalachia represent ‘sky islands’ due to their physical isolation, and they host numerous endemic species in other taxa. We surveyed globular Collembola through COI metabarcoding, assessing geographic and genetic diversity across localities and species. Intraspecific diversity in globular Collembola was extremely high, suggesting that considerable cryptic speciation has occurred. While we were able to associate morphospecies with described species in most of the major families in the region (Dicyrtomidae, Katiannidae, Sminthuridae, and Sminthurididae), other families (Neelidae, and Arrhopalitidae) are in more pressing need of taxonomic revision before species identities can be confirmed. Due to poor representation in databases, and high intraspecific variability, no identifications were accomplished through comparison with available DNA barcodes.

Assessing a megadiverse but poorly known community of fishes in a tropical mangrove estuary through environmental DNA (eDNA) metabarcoding

Biodiversity surveys are crucial for monitoring the status of threatened aquatic ecosystems, such as tropical estuaries and mangroves. Conventional monitoring methods are intrusive, time-consuming, substantially expensive, and often provide only rough estimates in complex habitats. An advanced monitoring approach, environmental DNA (eDNA) metabarcoding, is promising, although only few applications in tropical mangrove estuaries have been reported. In this study, we explore the advantages and limitations of an eDNA metabarcoding survey on the fish community of the Merbok Estuary (Peninsular Malaysia). COI and 12S eDNA metabarcoding assays collectively detected 178 species from 127 genera, 68 families, and 25 orders. Using this approach, significantly more species have been detected in the Merbok Estuary over the past decade (2010–2019) than in conventional surveys, including several species of conservation importance. However, we highlight three limitations: (1) in the absence of a comprehensive reference database the identities of several species are unresolved; (2) some of the previously documented specimen-based diversity was not captured by the current method, perhaps as a consequence of PCR primer specificity, and (3) the detection of non-resident species—stenohaline freshwater taxa (e.g., cyprinids, channids, osphronemids) and marine coral reef taxa (e.g., holocentrids, some syngnathids and sharks), not known to frequent estuaries, leading to the supposition that their DNA have drifted into the estuary through water movements. The community analysis revealed that fish diversity along the Merbok Estuary is not homogenous, with the upstream more diverse than further downstream. This could be due to the different landscapes or degree of anthropogenic influences along the estuary. In summary, we demonstrated the practicality of eDNA metabarcoding in assessing fish community and structure within a complex and rich tropical environment within a short sampling period. However, some limitations need to be considered and addressed to fully exploit the efficacy of this approach.

Assessment of short-term spatio-temporal variability in the structure of mesozooplankton communities integrating microscopy and multigene high-throughput sequencing

Variability in composition and structure of the mesozooplankton communities in the Bay of Malaga (SW Mediterranean) were characterized during a 26 h cycle using an integrative taxonomic approach. We combined microscopic identification of organisms, with metabarcoding for the genes of the mitochondrial DNA COI and the V9 hypervariable region of the ribosomal RNA 18S. Richness and diversity obtained by microscopy were higher than those measured with COI, as COI did not detect some phyla. COI however allowed for the identification to species level of several taxa that were left at higher taxonomic rank under the microscope. 18S detected a wider range of taxa than COI and microscopy, although with lower taxonomic resolution. Differences between coastal-night and shelf-day zooplankton communities structure were detected by both microscopy and metabarcoding. The combination of these two approaches increased the known copepod species in the SW Mediterranean Sea by 9%. An integrative approach combining morphology and COI metabarcoding is proposed to further facilitate mesozooplankton biodiversity studies.

Genomics of Serrasalmidae teleosts through the lens of microbiome fingerprinting.

Associations between host genotype and host-associated microbiomes have been shown in a variety of animal clades, but studies on teleosts mostly show weak associations. Our study aimed to explore these relationships in four sympatric Serrasalmidae (i.e., piranha) teleosts from an Amazonian lake, using data sets from the hosts genomes (single nucleotide polymorphisms from genotyping by sequencing), skin and gut microbiomes (16S rRNA gene metataxonomics) and diets (COI metabarcoding) from the same fish individuals. First, we investigated whether there were significant covariations of microbiome and fish genotypes at the inter- and intraspecific levels. We also assessed the extent of covariation between Serrasalmidae diet and microbiome, to isolate genotypic from dietary effects on community structure. We observed a significant covariation of skin microbiomes and host genotypes at interspecific (R2 = 24.4%) and intraspecific (R2 = 6.2%) levels, whereas gut microbiomes correlated poorly with host genotypes. Serrasalmidae diet composition was significantly correlated to fish genotype only at the interspecific level (R2 = 5.4%), but did not covary with gut microbiome composition (Mantel R= -.04). Second, we investigated whether the study of interspecific differentiation could benefit from considering host-associated microbial communities in addition to host genotypes. By using a nonmetric multidimensional scaling (NMDS) ordination-based approach, we observed that ordinations from skin- and gut species-specific bacterial biomarkers identified through a random forest algorithm could significantly increase the average interspecific differentiation detected through host genotype data alone. Although future studies encompassing additional species and environments are needed, our results suggest Serrasalmidae microbiomes could constitute an insightful trait to be considered when studying the interspecific differences between members of this clade.

Multi-marker DNA metabarcoding detects suites of environmental gradients from an urban harbour

There is increasing need for biodiversity monitoring, especially in places where potential anthropogenic disturbance may significantly impact ecosystem health. We employed a combination of traditional morphological and bulk macroinvertebrate metabarcoding analyses to benthic samples collected from Toronto Harbour (Ontario, Canada) to compare taxonomic and functional diversity of macroinvertebrates and their responses to environmental gradients. At the species rank, sites assessed using COI metabarcoding showed more variation than sites assessed using morphological methods. Depending on the assessment method, we detected gradients in magnesium (morphological taxa), ammonia (morphological taxa, COI sequence variants), pH (18S sequence variants) as well as gradients in contaminants such as metals (COI & 18S sequence variants) and organochlorines (COI sequence variants). Observed responses to contaminants such as aromatic hydrocarbons and metals align with known patchy distributions in harbour sediments. We determined that the morphological approach may limit the detection of macroinvertebrate responses to lake environmental conditions due to the effort needed to obtain fine level taxonomic assignments necessary to investigate responses. DNA metabarcoding, however, need not be limited to macroinvertebrates, can be automated, and taxonomic assignments are associated with a certain level of accuracy from sequence variants to named taxonomic groups. The capacity to detect change using a scalable approach such as metabarcoding is critical for addressing challenges associated with biodiversity monitoring and ecological investigations.

COI Metabarcoding Provides Insights into the Highly Diverse Diet of a Generalist Salamander, Salamandra salamandra (Caudata: Salamandridae)

DNA metabarcoding has proven to be an accessible, cost-effective, and non-invasive tool for dietary analysis of predators in situ. Although DNA metabarcoding provides numerous benefits in characterizing diet—such as detecting prey animals that are difficult to visually identify—this method has seen limited application in amphibian species. Here, we used DNA metabarcoding to characterize the diet of fire salamanders (Salamandra salamandra) (Linnaeus, 1758) in three distinct regions across the northwestern Iberian Peninsula. To test the efficiency of COI-based metabarcoding in determining salamanders’ diet diversity, we compared our COI-based results with results from traditional diet studies from neighboring and distant populations, as well as with recent findings obtained in a DNA metabarcoding study using 18S. Two COI primers were used in combination to investigate the potential impact of primer bias in prey detection. Our COI metabarcoding approach increased taxonomic resolution and supported a generalist diet in S. salamandra. Between primers, there were no significant differences in the diversity and richness of prey detected. We observed differences in the prevalence of prey identified between sampling regions both in our study and in other studies of S. salamandra diet. This COI metabarcoding study provides recommendations and resources for subsequent research using DNA metabarcoding to study amphibian diets.

Coming of age for COI metabarcoding of whole organism community DNA: Towards bioinformatic harmonisation

Metabarcoding of DNA extracted from community samples of whole organisms (whole organism community DNA, wocDNA) is increasingly being applied to terrestrial, marine and freshwater metazoan communities to provide rapid, accurate and high resolution data for novel molecular ecology research. The growth of this field has been accompanied by considerable development that builds on microbial metabarcoding methods to develop appropriate and efficient sampling and laboratory protocols for whole organism metazoan communities. However, considerably less attention has focused on ensuring bioinformatic methods are adapted and applied comprehensively in wocDNA metabarcoding. In this study we examined over 600 papers and identified 111 studies that performed COI metabarcoding of wocDNA. We then systematically reviewed the bioinformatic methods employed by these papers to identify the state‐of‐the‐art. Our results show that the increasing use of wocDNA COI metabarcoding for metazoan diversity is characterised by a clear absence of bioinformatic harmonisation, and the temporal trends show little change in this situation. The reviewed literature showed (i) high heterogeneity across pipelines, tasks and tools used, (ii) limited or no adaptation of bioinformatic procedures to the nature of the COI fragment, and (iii) a worrying underreporting of tasks, software and parameters. Based upon these findings we propose a set of recommendations that we think the metabarcoding community should consider to ensure that bioinformatic methods are appropriate, comprehensive and comparable. We believe that adhering to these recommendations will improve the long‐term integrative potential of wocDNA COI metabarcoding for biodiversity science.

EDNA reveals widespread presence of marine fish in the Panama Canal

Shipping corridors can be hotspots for biological invasions as they connect the world’s oceans and dissolve dispersal barriers between these aquatic systems. As a consequence, multiple opportunities for biotic exchange arise and the resulting establishment of non-native species often causes adverse ecological and economic impacts. In this study, a combined effort of traditional gillnetting and eDNA-based surveys was implemented to characterize the fish community of the Panama Canal, which is a key region for biotic exchange as it connects the Pacific and Atlantic Oceans in Central America. The Canal was recently expanded and new lock systems installed potentially providing novel opportunities for fish to enter the Canal. Using COI metabarcoding, we detected a total of 142 taxa, including thirteen potentially new records for the freshwater part of the Canal. Furthermore, we found evidence for the presence of 16 Atlantic and 10 Pacific non-native marine fish inside different sections of the Canal. Identifications based on molecular data did not cover all species caught with gillnets, but generally provided a more complete image of the fish fauna. Diversity indices based on eDNA surveys revealed significant differences across different sections of the Canal reflecting in part the prevailing environmental conditions. The observed changes in the fish community may be attributed to the recent expansion of the lock systems, which facilitates species migration directly by enlarging the canal entrances on either side of the Isthmus and indirectly by allowing more salt water to enter the Canal and thus increasing the overall salinity. Given the potential ecological and socio-economic consequences of non-native species crossing the Canal and establishing in the ocean basins on either end, it seems advisable to evaluate limiting salt water intrusion as well as implementing additional barriers to species dispersal (acoustic/electric/chemical deterrents) as a precautionary measure.

The limited spatial scale of dispersal in soil arthropods revealed with whole-community haplotype-level metabarcoding.

Soil arthropod communities are highly diverse and critical for ecosystem functioning. However, our knowledge of spatial structure and the underlying processes of community assembly are scarce, hampered by limited empirical data on species diversity and turnover. We implement a high-throughput sequencing approach to generate comparative data for thousands of arthropods at three hierarchical levels: genetic, species and supra-specific lineages. A joint analysis of the spatial arrangement across these levels can reveal the predominant processes driving the variation in biological assemblages at the local scale. This multihierarchical approach was performed using haplotype-level COI metabarcoding of entire communities of mites, springtails and beetles from three Iberian mountain regions. Tens of thousands of specimens were extracted from deep and superficial soil layers and produced comparative phylogeographic data for >1,000 codistributed species and nearly 3,000 haplotypes. Local assemblage composition differed greatly between grasslands and forests and, within each habitat, showed strong spatial structure and high endemicity. Distance decay was high at all levels, even at the scale of a few kilometres or less. The local distance decay patterns were self-similar for the haplotypes and higher hierarchical entities, and this fractal structure was similar in all regions, suggesting that uniform processes of limited dispersal determine local-scale community assembly. Our results from whole-community metabarcoding provide insight into how dispersal limitations constrain mesofauna community structure within local spatial settings over evolutionary timescales. If generalized across wider areas, the high turnover and endemicity in the soil locally may indicate extremely high richness globally, challenging our current estimations of total arthropod diversity on Earth.

Putting COI Metabarcoding in Context: The Utility of Exact Sequence Variants (ESVs) in Biodiversity Analysis

DNA barcoding and metabarcoding are techniques that focus on signature genomic regions that in theory provide species level resolution, but in practice this is not always possible. We place animal-focused COI metabarcoding in context with respect to the use of marker gene sequencing in microbial and fungal ecology. We focus on three specific aspects of metabarcodes: 1) the process of metabarcode sequence clustering, 2) how metabarcode cluster types affect the results of biodiversity analyses, and 3) we discuss the current state of reference sequence databases used for metabarcode identification. Using examples from the arthropod COI metabarcode literature, we show that exact sequence variants (ESVs) detect more unique taxa than operational taxonomic units (OTUs) but with similar patterns in taxonomic resolution. We also show that the difference between ordinations based on ESVs or OTUs recover similar groupings. We compile a list of reference sequence databases useful for multi-marker metabarcoding and present a list of reference sequence databases specifically formatted for use with a naive Bayesian classifier for rigorous metabarcode taxonomic assignments. Sophisticated tools and reference databases are available for analyzing COI sequences and these compare favorably with those available for other metabarcode markers such as the ribosomal RNA genes used to target microbes and fungi.

Variations in terrestrial arthropod DNA metabarcoding methods recovers robust beta diversity but variable richness and site indicators

Terrestrial arthropod fauna have been suggested as a key indicator of ecological integrity in forest systems. Because phenotypic identification is expert-limited, a shift towards DNA metabarcoding could improve scalability and democratize the use of forest floor arthropods for biomonitoring applications. The objective of this study was to establish the level of field sampling and DNA extraction replication needed for arthropod biodiversity assessments from soil. Processing 15 individually collected soil samples recovered significantly higher median richness (488–614 sequence variants) than pooling the same number of samples (165–191 sequence variants) prior to DNA extraction, and we found no significant richness differences when using 1 or 3 pooled DNA extractions. Beta diversity was robust to changes in methodological regimes. Though our ability to identify taxa to species rank was limited, we were able to use arthropod COI metabarcodes from forest soil to assess richness, distinguish among sites, and recover site indicators based on unnamed exact sequence variants. Our results highlight the need to continue DNA barcoding local taxa during COI metabarcoding studies to help build reference databases. All together, these sampling considerations support the use of soil arthropod COI metabarcoding as a scalable method for biomonitoring.