Stepwise implementation of high-throughput sequencing metabarcoding to estuarine macrobenthic communities

This work was supported by FEDER through POFC-COMPETE by national funds from ‘Fundação para a Ciência e a Tecnologia (FCT)’ in the scope of the grant FCOMP-01-0124-FEDER-015429 and also by the strategic programme UID/BIA/04050/2013 (POCI-01- 0145-FEDER-007569) also funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI). Jorge Lobo was supported by a PhD fellowship (SFRH/BD/69750/2010) from FCT.info:eu-repo/semantics/publishedVersio

DNA metabarcoding for high-throughput monitoring of estuarine macrobenthic communities

Morphology-based profling of benthic communities has been extensively applied to aquatic ecosystems’ health assessment. However, it remains a low-throughput, and sometimes ambiguous, procedure. Despite DNA metabarcoding has been applied to marine benthos, a comprehensive approach providing species-level identifcations for estuarine macrobenthos is still lacking. Here we report a combination of experimental and feld studies to assess the aptitude of COI metabarcoding to provide robust species-level identifcations for high-throughput monitoring of estuarine macrobenthos. To investigate the ability of metabarcoding to detect all species present in bulk DNA extracts, we contrived three phylogenetically diverse communities, and applied four diferent primer pairs to generate PCR products within the COI barcode region. Between 78–83% of the species in the contrived communities were recovered through HTS. Subsequently, we compared morphology and metabarcoding-based approaches to determine the species composition from four distinct estuarine sites. Our results indicate that species richness would be considerably underestimated if only morphological methods were used: globally 27 species identifed through morphology versus 61 detected by metabarcoding. Although further refnement is required to improve efciency and output of this approach, here we show the great aptitude of COI metabarcoding to provide high quality and auditable species identifcations in estuarine macrobenthos monitoring.This study has been funded by the project “Te NextSea: Next generation monitoring of coastal ecosystems in a scenario of global change” (operação NORTE-01-0145-FEDER-000032), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). JL was supported by a PhD fellowship (SFRH/BD/69750/2010) from FCT. Tis study had the fnancial support of Fundação para a Ciência e Tecnologia (FCT), through the strategic project UID/MAR/04292/2013 granted to MARE. Te authors would like to thank Stephanie Boilard (Biodiversity Institute of Ontario) for her support in the lab work.info:eu-repo/semantics/publishedVersio

Environmental Barcoding: A Next-Generation Sequencing Approach for Biomonitoring Applications Using River Benthos

Timely and accurate biodiversity analysis poses an ongoing challenge for the success of biomonitoring programs. Morphology-based identification of bioindicator taxa is time consuming, and rarely supports species-level resolution especially for immature life stages. Much work has been done in the past decade to develop alternative approaches for biodiversity analysis using DNA sequence-based approaches such as molecular phylogenetics and DNA barcoding. On-going assembly of DNA barcode reference libraries will provide the basis for a DNA-based identification system. The use of recently introduced next-generation sequencing (NGS) approaches in biodiversity science has the potential to further extend the application of DNA information for routine biomonitoring applications to an unprecedented scale. Here we demonstrate the feasibility of using 454 massively parallel pyrosequencing for species-level analysis of freshwater benthic macroinvertebrate taxa commonly used for biomonitoring. We designed our experiments in order to directly compare morphology-based, Sanger sequencing DNA barcoding, and next-generation environmental barcoding approaches. Our results show the ability of 454 pyrosequencing of mini-barcodes to accurately identify all species with more than 1% abundance in the pooled mixture. Although the approach failed to identify 6 rare species in the mixture, the presence of sequences from 9 species that were not represented by individuals in the mixture provides evidence that DNA based analysis may yet provide a valuable approach in finding rare species in bulk environmental samples. We further demonstrate the application of the environmental barcoding approach by comparing benthic macroinvertebrates from an urban region to those obtained from a conservation area. Although considerable effort will be required to robustly optimize NGS tools to identify species from bulk environmental samples, our results indicate the potential of an environmental barcoding approach for biomonitoring programs

DNA metabarcoding reveals metacommunity dynamics in a threatened boreal wetland wilderness

Too often, ecological monitoring studies are designed without understanding whether they have sufficient statistical power to detect changes beyond natural variability. The Peace–Athabasca Delta is North America’s largest inland delta, within a World Heritage area, and is currently threatened by human development. Using multispecies occupancy models we show that the wetland macroinvertebrate community is highly diverse, and spatial and temporal turnover are so high that composition is nearly random, emphasizing stochastic processes of assembly. Using DNA metabarcoding, our study detected more taxa, both overall and per sample, than traditional morphology-based sample processing, increasing our power to detect ecosystem change. Improving data quality and quantifying error are key to delivering effective monitoring and understanding the dynamic structure of the metacommunity.The complexity and natural variability of ecosystems present a challenge for reliable detection of change due to anthropogenic influences. This issue is exacerbated by necessary trade-offs that reduce the quality and resolution of survey data for assessments at large scales. The Peace–Athabasca Delta (PAD) is a large inland wetland complex in northern Alberta, Canada. Despite its geographic isolation, the PAD is threatened by encroachment of oil sands mining in the Athabasca watershed and hydroelectric dams in the Peace watershed. Methods capable of reliably detecting changes in ecosystem health are needed to evaluate and manage risks. Between 2011 and 2016, aquatic macroinvertebrates were sampled across a gradient of wetland flood frequency, applying both microscope-based morphological identification and DNA metabarcoding. By using multispecies occupancy models, we demonstrate that DNA metabarcoding detected a much broader range of taxa and more taxa per sample compared to traditional morphological identification and was essential to identifying significant responses to flood and thermal regimes. We show that family-level occupancy masks high variation among genera and quantify the bias of barcoding primers on the probability of detection in a natural community. Interestingly, patterns of community assembly were nearly random, suggesting a strong role of stochasticity in the dynamics of the metacommunity. This variability seriously compromises effective monitoring at local scales but also reflects resilience to hydrological and thermal variability. Nevertheless, simulations showed the greater efficiency of metabarcoding, particularly at a finer taxonomic resolution, provided the statistical power needed to detect change at the landscape scale

High Throughput Automated Allele Frequency Estimation by Pyrosequencing

Pyrosequencing is a DNA sequencing method based on the principle of sequencing-by-synthesis and pyrophosphate detection through a series of enzymatic reactions. This bioluminometric, real-time DNA sequencing technique offers unique applications that are cost-effective and user-friendly. In this study, we have combined a number of methods to develop an accurate, robust and cost efficient method to determine allele frequencies in large populations for association studies. The assay offers the advantage of minimal systemic sampling errors, uses a general biotin amplification approach, and replaces dTTP for dATP-apha-thio to avoid non-uniform higher peaks in order to increase accuracy. We demonstrate that this newly developed assay is a robust, cost-effective, accurate and reproducible approach for large-scale genotyping of DNA pools. We also discuss potential improvements of the software for more accurate allele frequency analysis

Pyrosequencing for Mini-Barcoding of Fresh and Old Museum Specimens

DNA barcoding is an effective approach for species identification and for discovery of new and/or cryptic species. Sanger sequencing technology is the method of choice for obtaining standard 650 bp cytochrome c oxidase subunit I (COI) barcodes. However, DNA degradation/fragmentation makes it difficult to obtain a full-length barcode from old specimens. Mini-barcodes of 130 bp from the standard barcode region have been shown to be effective for accurate identification in many animal groups and may be readily obtained from museum samples. Here we demonstrate the application of an alternative sequencing technology, the four-enzymes single-specimen pyrosequencing, in rapid, cost-effective mini-barcode analysis. We were able to generate sequences of up to 100 bp from mini-barcode fragments of COI in 135 fresh and 50 old Lepidoptera specimens (ranging from 53–97 year-old). The sequences obtained using pyrosequencing were of high quality and we were able to robustly match all the tested pyro-sequenced samples to their respective Sanger-sequenced standard barcode sequences, where available. Simplicity of the protocol and instrumentation coupled with higher speed and lower cost per sequence than Sanger sequencing makes this approach potentially useful in efforts to link standard barcode sequences from unidentified specimens to known museum specimens with only short DNA fragments

Interspecific competition in bats and diet shifts in response to white‐nose syndrome

Since the introduction of white‐nose syndrome (WNS ) in North America, numerous species of bat have dwindled in numbers. These declines observed are often species‐specific and thus provides opportunity for a natural experiment to test for shifts in diet through relaxed resource partitioning in bat communities post‐introduction of WNS . Acoustic monitoring at locations in Southern Ontario pre‐ (2009–2011) and post‐WNS (2012–2014) introduction showed an increase in activity of big brown bats (Eptesicus fuscus ) corresponding to a decline in the activity of little brown bats (Myotis lucifugus ). Next‐generation sequencing of bat stomachs and guano in Southern Ontario before and after WNS allowed for the characterization of diet changes of these species. Post‐WNS , E. fuscus consumed a wider breadth of prey and many of the insect species once consumed by M. lucifugus , including several pest insects. These results suggest that the introduction of WNS has resulted in relaxed interspecific competition within these bat communities in Southern Ontario

Interspecific competition in bats and diet shifts in response to white‐nose syndrome

Since the introduction of white‐nose syndrome (WNS ) in North America, numerous species of bat have dwindled in numbers. These declines observed are often species‐specific and thus provides opportunity for a natural experiment to test for shifts in diet through relaxed resource partitioning in bat communities post‐introduction of WNS . Acoustic monitoring at locations in Southern Ontario pre‐ (2009–2011) and post‐WNS (2012–2014) introduction showed an increase in activity of big brown bats (Eptesicus fuscus ) corresponding to a decline in the activity of little brown bats (Myotis lucifugus ). Next‐generation sequencing of bat stomachs and guano in Southern Ontario before and after WNS allowed for the characterization of diet changes of these species. Post‐WNS , E. fuscus consumed a wider breadth of prey and many of the insect species once consumed by M. lucifugus , including several pest insects. These results suggest that the introduction of WNS has resulted in relaxed interspecific competition within these bat communities in Southern Ontario

Data from: Discrimination of grasshopper (Orthoptera: Acrididae) diet and niche overlap using next-generation sequencing of gut contents

Species of grasshopper have been divided into three diet classifications based on mandible morphology: forbivorous (specialist on forbs), graminivorous (specialist on grasses), and mixed feeding (broad-scale generalists). For example, Melanoplus bivittatus and Dissosteira carolina are presumed to be broad-scale generalists, Chortophaga viridifasciata is a specialist on grasses, and Melanoplus femurrubrum is a specialist on forbs. These classifications, however, have not been verified in the wild. Multiple specimens of these four species were collected, and diet analysis was performed using DNA metabarcoding of the gut contents. The rbcLa gene region was amplified and sequenced using Illumina MiSeq sequencing. Levins' measure and the Shannon–Wiener measure of niche breadth were calculated using family-level identifications and Morisita's measure of niche overlap was calculated using operational taxonomic units (OTUs). Gut contents confirm both D. carolina and M. bivittatus as generalists and C. viridifasciata as a specialist on grasses. For M. femurrubrum, a high niche breadth was observed and species of grasses were identified in the gut as well as forbs. Niche overlap values did not follow predicted patterns, however, the low values suggest low competition between these species