Biodiversity Assessment Tool Research Articles

Comparative analysis of bottom trawl and nanopore sequencing in fish biodiversity assessment: The sylt outer reef example

The assessment of fish diversity is crucial for effective conservation and management strategies, especially in ecologically sensitive regions such as marine protected areas. This study contrasts the effectiveness of environmental DNA (eDNA) metabarcoding analysis employing Nanopore technology with compare beam trawl surveys at the Sylt Outer Reef, a Natura 2000 site in the North Sea, Germany.Out of the 17 fish species caught in a bottom trawl (using a 3m beam trawl), 14 were also identified through eDNA extracted from water samples. The three species not detected in the eDNA results were absent because they lacked representation in public DNA databases. The eDNA method detected twice as many fish species as the beam trawl, totalling 36 species, of which 14 were also detected by the trawl. Additionally, the selection of primers (Mifish) facilitated the identification of one marine mammal species, the harbour porpoise.In conclusion, the findings underscore the potential of eDNA coupled with MinION sequencing (Long read technology) as a robust tool for biodiversity assessment, surpassing traditional methods in detecting species richness.

Assessing the Impacts of Changing Connectivity of Hydropower Dams on the Distribution of Fish Species in the 3S Rivers, a Tributary of the Lower Mekong

Hydropower plants (HPPs) create barriers across rivers and fragment aquatic ecosystems, river reaches and habitats. The reservoirs they create slow the flowing water and convert the riverine into lacustrine ecosystems. The barriers created by HPPs interrupt the seasonal migrations of many fish species, while the reservoirs drive away fish species that are dependent on flowing water habitats. This paper assesses the distribution of fish species in the 3S rivers—Sekong, Sesan and Sre Pok, in Cambodia, Laos and Viet Nam—using IUCN Red List-assessed species distribution by HydroBasin Level 8 from the freshwater reports of the Integrated Biodiversity Assessment Tool (IBAT) and their connectivity with the Mekong. There are currently 61 commissioned dams in the 3S basins and a further 2 under construction, 23 of which are larger than the 30 MW installed capacity. A further 24 HPPs are proposed or planned in these basins. The changes in connectivity caused by the dams are measured by adapting the River Class Connectivity Index (RCICLASS); the original connectivity of the 3S basin taking into account the two major waterfalls in the Sesan and Sre Pok rivers was estimated at 80.9%. With existing dams, the connectivity has been reduced to 23.5%, and with all planned dams, it is reduced further to 10.9%. The resulting re-distribution of fish species occurring throughout the 3S basins is explored, by focusing on migratory guilds and threatened and endemic fish species. With all dams built, it is predicted that the total numbers of species in HydroBasins above the dams will be reduced by 40–50%. The Threatened Species Index is estimated to fall from over 30 near the confluence of the three rivers to less than 10 above the lowest dams on the 3S rivers. The analysis demonstrates how widely available global and regional datasets can be used to assess the impacts of dams on fish biodiversity in this region.

Exploring the spectral variation hypothesis for α- and β-diversity: a comparison of open vegetation and forests

Airborne hyperspectral imaging holds great promise for estimating plant diversity and composition, given its unprecedented combination of aerial coverage, spatial resolution, and spectral detail. Recently, there has been renewed attention toward the spectral variation hypothesis (SVH), which predicts that higher spectral variation is correlated with greater plant diversity. While several studies have highlighted methodological challenges involved with the SVH, there is little consensus about when it yields strong predictions of taxonomic, functional, and phylogenetic diversity. In part, this may be because prior studies have not explicitly considered how underlying environmental gradients drive changes in spectral and species composition. In this study, we tested the SVH separately in open vegetation (i.e. grasses and shrubs) and in forests at five sites across Canada. Generalized additive models revealed that spectral diversity was a better predictor of functional α-diversity than of taxonomic or phylogenetic α-diversity in both vegetation types. Mantel tests and Procrustes analyses revealed weak to moderate associations between spectral and plant β-diversity and composition in open vegetation, and moderate associations in forests. The better fit in forests appeared to be influenced by the presence of an elevational gradient and associated species turnover (from deciduous to coniferous trees); we observed weaker relationships when examining only a subset of this gradient. We suggest that the high variability in the strength of associations between plant and spectral diversity reported to date might be affected by the presence of environmental gradients. Finally, we found that different wavelength bands contributed to spectral α-diversity in open vegetation vs. forests, suggesting different spectral features are important for different vegetation types. In conclusion, spectral diversity is a potentially powerful tool for biodiversity assessment, but it requires a context-specific approach.

The First International eDNA Workshop in Hong Kong: A beginner's guide for the next‐generation eDNA researcher

AbstractThe field of environmental DNA (eDNA) has rapidly advanced in recent years, providing a non‐invasive and time‐saving method for assessing biodiversity. The First International Environmental DNA (eDNA) Workshop in Hong Kong was held from 16 to 27 October 2023 and provided early career professionals with hands‐on training and collaboration opportunities in eDNA research. With support from The Croucher Foundation Limited (Hong Kong), the workshop covered all stages of an eDNA‐based research project, including study design, field sampling, eDNA extraction, library preparation, bioinformatics, statistical data analysis, and ethics in scientific research. Participants gained insights into the principles and practical applications of eDNA technology, emphasizing the importance of careful experimental design and data interpretation. The workshop also highlighted the need for standardized protocols, comprehensive and local DNA reference databases, and careful selection of primer sets to overcome current issues and limitations. Workshop participants expressed enthusiasm for the potential of eDNA metabarcoding as a valuable tool for ecological monitoring, biodiversity assessment, and conservation decision‐making. The future of eDNA research looks promising overall, with continued advancements in technology, collaboration among researchers, and the integration of eDNA into large‐scale ecological monitoring. Future iterations of the Hong Kong International eDNA workshop will continue to provide hands‐on training and collaboration opportunities for early career professionals interested in eDNA research, focusing on addressing current limitations and challenges in the field.

Using butterfly survey data to model habitat associations in urban developments

Abstract One approach for measuring the potential biodiversity in new urban construction projects is through ecological models that predict how wildlife will respond. For the United Kingdom, such models have only been developed for birds, but to maximise the extent to which models represent overall biodiversity, species from different indicator groups must be considered. Here, we assess this possibility for butterflies by combining citizen science survey data with high‐resolution digital maps. We derive detailed characteristics of urban landscapes around survey sites using previously established methods and quantify their relationship to counts of adults of 18 butterfly species in urban and peri‐urban settings. Higher butterfly counts were found when traversing urban sites with larger areas of semi‐natural grassland, other managed greenspaces and adjacent arable land. Most of the butterfly community were found to have negative relationships with highly built‐up or fragmented landscapes. We found high species‐specificity for different details of urban form, particularly in habitat elements such as gardens, vegetation around railways and grass verges. Policy implications. Improving biodiversity is now part of legislation governing new construction projects from England and Wales. However, predicting quantitative changes from hypothetical land‐use modifications remains challenging. Our models provide the foundation through which butterfly abundance could be integrated into an urban biodiversity assessment tool, providing species‐ and community‐level statistics to non‐specialists from the urban planning and design sector. This would allow them to hone configurations for built surfaces, private gardens, greenspaces and wildlife areas and assess their capacity to provide residents with the intended access to nature.

EDNA metabarcoding as a means to track distributions of different fish species in a protected area

AbstractIn Lar National Park (Caspian Sea basin, Iran), the Caspian trout (Salmo caspius) population faces different threats, including introduced fish species. Due to the harsh environmental conditions and limited accessibility, monitoring of fish species via conventional approaches proves difficult. Hence, environmental DNA metabarcoding may prove an appropriate tool for monitoring fishes within the park. Environmental DNA samples from eight stream sites in the National Park were sequenced via metabarcoding of the 12S rRNA gene, and the species identified via eDNA metabarcoding were compared to the results of electrofishing performed at the same localities on the same day. No significant difference in the number of Caspian Sea trout DNA sequence reads was detected among the collection sites (p > 0.05). The highest number of reads was detected in Dalichay Stream, but the highest population density determined via electrofishing was in Siahpalas Stream. The discrepancy between the eDNA read count and trout population density, as well as the limited sampling scheme within this study, limit our ability to provide a robust conclusion about the application of environmental DNA metabarcoding for assessment of fish density in Lar National Park. Environmental DNA metabarcoding detected more species than electrofishing, but no significant differences in the composition of the local fish community were observed. Introduced fish species were all observed or detected in Siahpalas Stream, which is characterized by high water temperature, muddy substrate, and lower flow rate. A significant effect of flow rate and total dissolved solids on the presence of introduced fish species (p = 0.02) and of flow rate alone on relative abundance of introduced fish species (p = 0.03) was detected. To standardize the application of eDNA metabarcoding as a biodiversity assessment tool in Lar National Park, future studies should characterize the parameters that affect eDNA persistence and detectability in the system.

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.

Species delimitation and molecular phylogeny of the grasshopper subfamily Gomphocerinae (Orthoptera: Acrididae) from Algeria based on mitochondrial and nuclear DNA markers

Gomphocerinae grasshoppers (Orthoptera: Acrididae) are generally characterised by their polymorphism and cryptic diversity, which can be confusing when relying on morphological identification alone. DNA taxonomy serves as a powerful molecular tool for species identification and biodiversity assessment. In the context of zoogeography and the biodiversity conservation of animal resources in ecosystems, DNA barcoding data for Algerian Gomphocerinae fauna remains limited in global databases, despite their agroeconomic and environmental importance. Therefore, in this study, we collected various species of this subfamily from the Biskra region in Algeria and conducted DNA barcoding analysis, employing different molecular species delimitation methods (the automatic barcode gap discovery, assemble species by automatic partitioning, single-threshold general mixed Yule coalescent model, Bayesian Poisson tree process and multi-rate Poisson tree process methods), as well as phylogenetic analyses (maximum-likelihood and Bayesian inference) based on two molecular markers (COI and ITS). The results of the 163 newly generated sequences demonstrated that DNA barcoding technology is highly efficient and valuable for species identification in the subfamily Gomphocerinae, showing strong congruence with morphological evidence for 12 species, including eight species that were sequenced for the first time. This study also reported a new record of the genus Stenohippus Uvarov, 1926 in Algeria, comprising two species. Additionally, taxonomic revision allowed the species Dociostaurus biskrensis Moussi & Petit, 2014, to be transferred to the genus Stenohippus. Molecular tree analyses revealed the phylogenetic positions of the newly sequenced species within the subfamily Gomphocerinae and provided insights into their evolutionary relationships. These new data serve as a starting point for future research in other geographical areas, enabling a better understanding of the biodiversity of this insect group in Algeria.

A frequency-dependent acoustic diversity index: A revision to a classic acoustic index for soundscape ecological research

In recent years, passive acoustic monitoring (PAM) has been widely applied in the field of soundscape ecology. Meanwhile, acoustic indices, as an important tool for rapid biodiversity assessment (RBA), have also attracted increasing attention. However, considerable evidence demonstrates that existing acoustic indices are susceptible to noise, which poses a challenge for wider applications of acoustic indices. In this work, we firstly provide insights into how noise affects the widely used acoustic diversity index (ADI), revealing that the global threshold scheme (i.e., −50 dBFS for all time–frequency bins) to generate the binary spectrogram (spectrogram containing time–frequency bins denoted as ‘1′ with power above the threshold and ‘0′ below) is the key factor that makes ADI sensitive to noise. To address this problem, we propose a revised ADI version named frequency-dependent acoustic diversity index (FADI), which employs a new threshold scheme to reduce noise impacts on its values. Specifically, considering that the noise floor along frequency axis usually varies in practice, i.e., a colored noise rather than a white noise exists in field recordings, floating thresholds adapted to the noise level at each frequency bin were employed and further incorporated with the global threshold of ADI. Controlled computational experiments using field recordings were conducted where manipulated noise levels were introduced as different signal-to-noise ratios (SNR). It was shown that the proposed FADI was significantly robust to noise within a much wider SNR range as compared with ADI. Furthermore, a singular example using a real-world recording also verified its effectiveness and highlighted the promising potential of FADI in applications of PAM.

Environmental DNA detects biodiversity and ecological features of phytoplankton communities in Mediterranean transitional waters

Climate changes and anthropogenic pressures are causing a biodiversity decline in terms of species number and genetic diversity, reducing the adaptability and evolvability of natural communities. Transitional water ecosystems are more sensitive to habitat reduction and degradation and, thus, are more exposed to biodiversity declines requiring biodiversity monitoring programs for their conservation. Environmental DNA (eDNA) metabarcoding represents a high-throughput tool for biodiversity assessment that is facilitating data collection for biodiversity monitoring. In this study, we applied, for the first time, eDNA metabarcoding in a Mediterranean coastal lagoon to assess the ecological features of eukaryotic phytoplankton communities. We sampled water in seven different lagoon sites and amplified the extracted DNA with primers targeting the variable region 4 (V4) of the 18S rRNA gene marker. The results demonstrated the validity of eDNA studies to provide insights into lagoon phytoplankton composition, establish the structure and spatial variation of phytoplankton communities, and evaluate its correlation to abiotic factors. Finally, the genetic distances analysis suggests that the different spatial distribution of OTUs, at least for the Tetraselmis genus, reflects the genetic background.

Biodiversity Conservation in Mining Landscapes: a Systematic Review of Assessment Approaches in the Philippines

Mining is an important sector of the Philippine economy, but it often results in negative impacts on biodiversity. To mitigate these impacts, it is crucial to assess biodiversity in mining areas and integrate conservation efforts into mining operations. As part of the national policy guidelines on biodiversity compliance for mining companies, this study has conducted a systematic review to examine the various biodiversity assessment methods, tools, sampling designs, diversity parameters, values, and indices used in the Philippines' mining sites. Search engines and research databases were utilized in identifying diversity assessment-related research. Based on the eligibility criteria, only 25 of the 100 papers downloaded and two project terminal reports were eligible and considered in the review. It was found that authors commonly used transect line and quadrat methods for floral studies and transect line, mist netting, point count, and opportunistic sampling for faunal studies. Species abundance, relative abundance, dominance, frequency, relative frequency, density, relative density, percent cover, and importance value were the most frequently assessed biodiversity parameters, whereas the Shannon-Wiener diversity index, Simpson index, species richness, and evenness were the most commonly used indices. Endemism, economic importance, invasiveness, and conservation status were additional factors evaluated. Overall, this review provides an overview of the various biodiversity assessment methods used in the country's mining areas and offers guidance for future assessments in other mining landscapes. The limited number of studies related to biodiversity assessment in mining areas in the Philippines over the last decade highlights the urgent need for more research in this field.

Identification, prioritization, and management of biodiversity hot specks: a case study of Western Ghats of Maharashtra, India

The Western Ghats are globally recognized as a hotspot of rich, endemic, and threatened biodiversity. Within this hotspot of biological diversity, there are islands of natural landscapes that can be termed as ‘hot specks’. These hot specks require careful prioritization and specific management strategies as they vary in objectives and ownership. Conserving hot specks of biodiversity is of great relevance because creating new protected areas with wildlife corridors between them has become relatively impossible in the present context of intensive land-use change in this rapidly developing region. Management strategies, however, must be based on scientific assessment and using a set of prioritization criteria for selecting the most appropriate forms of management. The conservation action plan for the Western Ghats has become a controversial issue based on the findings in the report submitted by the Western Ghats Expert Ecological Panel and the High Level Work Group on Western Ghats. In the present context of rapidly changing land-use patterns, economic development, forest fragmentation, isolation of habitats, linear intrusion, neo-urbanization and industrial growth are threats to the pristine nature of the ghats. Thus, there is an urgent need to identify, prioritize and manage the smaller fragments of biological importance within the larger ecologically sensitive landscape. A prioritization model for different types of hot specks is essential so that it can be easily replicated by training frontline forest staff, community-based organizations, Biodiversity Management Committees, and non-government organizations for implementing a strategy and action plans for the sites by using the Biological Diversity Act, 2002. Support of the local Biodiversity Management Committees and the State Biodiversity Board is essential for the conservation management of these biodiversity-rich sites. This study presents an innovative approach to prioritize areas outside the formally notified boundaries of the national parks and wildlife sanctuaries to assess the conservation value of hot specks of diversity through a rapid biodiversity assessment tool. This can lead to a rational conservation strategy that conservation planners and practitioners can use.

Maximizing sampling efficiency to detect differences in fish community composition using environmental DNA metabarcoding in subarctic fjords

AbstractEnvironmental DNA (eDNA) has gained popularity as a tool for ecosystem biomonitoring and biodiversity assessment. Although much progress has been made regarding laboratory and fieldwork protocols, the issue of sampling efficiency requires further investigation, particularly in three‐dimensional marine systems. This study focuses on fish community composition in marine ecosystems and aims to analyze the efficiency of sampling design given the sampling effort for distinguishing between different communities. We sampled three fjords in Northern Norway, taking samples along fjord transects and at three different depths, and amplified a fragment of the mitochondrial 12S rRNA gene of bony fishes using the MiFish primers. We evaluated the effect of (i) the number of sampling stations, (ii) samples' spatial distribution, and (iii) the data treatment approach (presence/absence versus semiquantitative) for maximizing the efficiency of eDNA metabarcoding sampling when inferring differences of fish community compositions between fjords. We found that the manner of data treatment strongly affected the minimum number of sampling stations required to detect differences among communities; because the semiquantitative approach retained some information about abundance of the underlying reads, it was the most efficient. Furthermore, we found little‐to‐no difference of fish communities in samples from intermediate depths when comparing vertical fish communities. Lastly, we found that the differences between fish communities at the surface were the highest across the horizontal distance and overall, samples ~30 km apart showed the highest variation in the horizontal distribution. Boosting sampling efficiency (reducing sampling effort without compromising ecological inferences) can significantly contribute to enhanced biodiversity management and efficient biomonitoring plans.

Unlocking the genetic code: Exploring the potential of DNA barcoding for biodiversity assessment

<abstract> <p>DNA barcoding is a crucial method for assessing and monitoring species diversity amidst escalating threats to global biodiversity. I explore DNA barcoding's potential as a robust and reliable tool for biodiversity assessment. It begins with a comprehensive review of existing literature, delving into the theoretical foundations, methodologies and applications of DNA barcoding. The suitability of various DNA regions, like the COI gene, as universal barcodes are extensively investigated. Additionally, the advantages and limitations of different DNA sequencing technologies and bioinformatics tools are evaluated within the context of DNA barcoding. To evaluate the efficacy of DNA barcoding, diverse ecosystems, including terrestrial, freshwater and marine habitats, are sampled. Extracted DNA from collected specimens undergoes amplification and sequencing of the target barcode region. Comparison of the obtained DNA sequences with reference databases allows for the identification and classification of the sampled organisms. Findings demonstrate that DNA barcoding accurately identifies species, even in cases where morphological identification proves challenging. Moreover, it sheds light on cryptic and endangered species, aiding conservation efforts. I also investigate patterns of genetic diversity and evolutionary relationships among different taxa through the analysis of genetic data. This research contributes to the growing knowledge on DNA barcoding and its applicability for biodiversity assessment. The advantages of this approach, such as speed, accuracy and cost-effectiveness, are highlighted, along with areas for improvement. By unlocking the genetic code, DNA barcoding enhances our understanding of biodiversity, supports conservation initiatives and informs evidence-based decision-making for the sustainable management of ecosystems.</p> </abstract>

Alpine freshwater fish biodiversity assessment: an inter-calibration test for metabarcoding method set up

The analysis of environmental DNA (eDNA) by high throughput sequencing (HTS) is proving to be a promising tool for freshwater fish biodiversity assessment in Europe within the Water Framework Directive (WFD, 2000/60/EC), especially for large rivers and lakes where current fish monitoring techniques have known shortcomings. These new biomonitoring methods based on eDNA show several advantages compared to classical morphological methods. The sampling procedures are easier and cheaper and eDNA metabarcoding is non-invasive and very sensitive, allowing for the detection of traces of DNA. However, eDNA metabarcoding methods need careful standardization to make the results of different surveys comparable. The aim of the EU project Eco-AlpsWater is to test and validate molecular biodiversity monitoring tools for aquatic ecosystems (i.e., eDNA metabarcoding) to improve the traditional WFD monitoring approaches in Alpine waterbodies. To this end, an inter-calibration test was performed using fish mock community samples containing either tissue-extracted DNA, eDNA collected from aquaculture tanks and eDNA samples collected from Lake Bourget (France). Samples were analysed using a DNA metabarcoding approach, relying on the amplification and HTS of a 12S rDNA marker, in two separate laboratories, to evaluate if different laboratory and bioinformatic protocols can provide a reliable and comparable description of the fish communities in both mock and natural samples. Our results highlight good replicability of the molecular laboratory protocols for HTS and good amplification success of selected primers, providing essential information concerning the taxonomic resolution of the 12S mitochondrial marker in describing the Alpine fish communities. Interestingly, different concentrations of species DNA in the mock samples were well represented by the relative DNA reads abundance. These tests confirm the reproducibility of eDNA metabarcoding analyses for the biomonitoring of freshwater fish inhabiting Alpine and peri-Alpine lakes and rivers.

Home is where the hollow is: Revealing vertebrate tree hollow user biodiversity with eDNA metabarcoding

AbstractTree hollows are essential for many vertebrate species, providing both nesting sites and shelter. Globally, old hollow‐bearing trees are in decline resulting in many dependent species being under threat. It is, therefore, imperative that vital hollow‐bearing trees are preserved, but it is logistically difficult to rapidly determine which hollows are being used and by which taxa. Here, we investigate the efficacy of eDNA metabarcoding as a survey tool for vertebrate hollow users. We compared the identity and richness of hollow‐inhabiting vertebrate taxa using eDNA metabarcoding of both sediment from the tree hollows, and material collected using roller swabs. Samples (n = 138) were collected from hollow‐bearing tuart trees (Eucalyptus gomphocephala; N = 28), within both an urban remnant and a relatively undisturbed forested area of South‐West Western Australia. We detected a wide range of vertebrate taxa, including cryptic species such as the brush‐tailed phascogale (Phascogale tapoatafa), while also providing ecologically informative data, such as hollow use by invasive Rainbow Lorikeet (Trichoglossus moluccanus) within the study areas. Our results showed variation in the species detected between methods, with the roller swab method detecting a greater number of species and a higher mean species richness per sample than hollow sediment did. The species detected from both methods did not perfectly overlap, highlighting the value of using multiple methods or substrates to detect a greater number of taxa. Our results suggest eDNA metabarcoding from tree hollow samples offers a sensitive and resource‐efficient method of monitoring vertebrate hollow users, if enough hollows are sampled. This provides not only a broad biodiversity assessment tool, but also an effective method for detecting taxa that may be elusive using other methods.

Holistic Impact Evaluation of Human Activities on the Coastal Fish Biodiversity in the Chinese Coastal Environment.

Ecological qualities and resources in coasts are threatened by various human activities, such as pollution and fishery. Impact evaluation of environmental stressors over a wide coastal stretch has been limited due to lack of efficient and standardizable biodiversity monitoring and assessment tools. Integrating environmental DNA (eDNA) and ecological traits, a holistic approach was developed to assess the impact of pollution and aquaculture on fish biodiversity in Chinese coastal areas. Taking the Yalujiang Estuary (YLJK) from the Yellow Sea and the Nan'ao Island Area (NAO) from the South China Sea as cases, the performance of the eDNA biomonitoring workflow was validated. First, the eDNA results of 22 sampling sites reached more than 85% of the asymptotes of species or ASVs in each area. A total of 115 fish species in both areas were detected and NAO was 1.8 times richer than YLJK using eDNA and the fish eDNA composition was consistent with the historical data. eDNA recovered distinct variations of fish sequence, taxonomic and functional diversity, and the corresponding trends following the offshore distance between the two areas. Fish sequence diversity was decreased primarily by estuarine pollution factors (chemical oxygen demand and zinc) in the YLJK. Compared with no breeding areas, lower fish sequence diversity was in breeding areas in the NAO. By integrating ecological traits, the eDNA approach offers promising opportunities for future fish biodiversity monitoring and assessment in national and global coastal environments.

Effects of sampling strategies and DNA extraction methods on eDNA metabarcoding: A case study of estuarine fish diversity monitoring

Environmental DNA (eDNA) integrated with metabarcoding is a promising and powerful tool for species composition and biodiversity assessment in aquatic ecosystems and is increasingly applied to evaluate fish diversity. To date, however, no standardized eDNA-based protocol has been established to monitor fish diversity. In this study, we investigated and compared two filtration methods and three DNA extraction methods using three filtration water volumes to determine a suitable approach for eDNA-based fish diversity monitoring in the Pearl River Estuary (PRE), a highly anthropogenically disturbed estuarine ecosystem. Compared to filtration-based precipitation, direct filtration was a more suitable method for eDNA metabarcoding in the PRE. The combined use of DNeasy Blood and Tissue Kit (BT) and traditional phenol/chloroform (PC) extraction produced higher DNA yields, amplicon sequence variants (ASVs), and Shannon diversity indices, and generated more homogeneous and consistent community composition among replicates. Compared to the other combined protocols, the PC and BT methods obtained better species detection, higher fish diversity, and greater consistency for the filtration water volumes of 1 000 and 2 000 mL, respectively. All eDNA metabarcoding protocols were more sensitive than bottom trawling in the PRE fish surveys and combining two techniques yielded greater taxonomic diversity. Furthermore, combining traditional methods with eDNA analysis enhanced accuracy. These results indicate that methodological decisions related to eDNA metabarcoding should be made with caution for fish community monitoring in estuarine ecosystems.

Environmental DNA metabarcoding as a tool for biodiversity assessment and monitoring: reconstructing established fish communities of north‐temperate lakes and rivers

AbstractAimTo evaluate the ability of precipitation‐based environmental DNA (eDNA) sample collection and mitochondrial 12S metabarcoding sequencing to reconstruct well‐studied fish communities in lakes and rivers. Specific objectives were to 1) determine correlations between eDNA species detections and known community composition based on conventional field sampling, 2) compare efficiency of eDNA to detect fish biodiversity among systems with variable morphologies and trophic states, and 3) determine if species habitat preferences predict eDNA detection.LocationUpper Great Lakes Region, North America.MethodsFish community composition was estimated for seven lakes and two Mississippi River navigation pools using sequence data from the mitochondrial 12S gene amplified from 10 to 50 water samples per waterbody collected in 50‐mL centrifuge tubes at a single time point. Environmental DNA (eDNA) was concentrated without filtration by centrifuging samples to reduce per‐sample handling time. Taxonomic detections from eDNA were compared to established community monitoring databases containing up to 40 years of sampling and a detailed habitat/substrate preference matrix to identify patterns of bias.ResultsMitochondrial 12S gene metabarcoding detected 15%–47% of the known species at each waterbody and 30%–76% of known genera. Non‐metric multidimensional scaling (NMDS) assessment of the community structure indicated that eDNA‐detected communities grouped in a similar pattern as known communities. Discriminant analysis of principal components indicated that there was a high degree of overlap in habitat/substrate preference of eDNA‐detected and eDNA‐undetected species suggesting limited habitat bias for eDNA sampling.Main conclusionsLarge numbers of small volume samples sequenced at the mitochondrial 12S gene can describe the coarse community structure of freshwater systems. However, additional conventional sampling and environmental DNA sampling may be necessary for a complete diversity census.

Assessment of the macroinvertebrate community of the Vjosa river through non-destructive DNA metabarcoding of preservative ethanol

Streams and rivers represent hotspots of biodiversity in their natural state. This biodiversity is declining worldwide due to pollution, exploitation and hydromorphological degradation of these systems. One of the last big, natural rivers in Europe is the Vjosa in the Balkan region. The catchment is characterized by natural flow dynamics, resulting in high habitat diversity and turnover, and hosts several sensitive and endemic species (e.g. Isoperla vjosae). DNA metabarcoding represents a promising approach to characterize this biodiversity but methodological drawbacks such as primer bias or incomplete reference databases limit the application, particularly in taxonomically underexplored regions. Here, we assessed stream biodiversity with a focus on macrozoobenthic (MZB) taxa via a non-destructive, voucher-preserving DNA metabarcoding protocol. In this approach, ethanol used for preservation of multi-habitat samples in the field was used as DNA template, allowing to retain the integrity of the original sample and further comparison of molecular and morphological taxa lists. Samples were taken in spring and autumn 2018 at 48 sites allocated over the Vjosa catchment. The preservative ethanol was filtered through 0.43 um nitrocellulose membranes from which DNA was extracted. Subsequently, a 421 bp fragment of the COI gene was amplified with the primer pair BF2/BR2 and Illumina sequenced. After filtering for sequences with similarity to reference entries >85%, 4,123 OTUs were obtained, of which 921 were identified as MZB taxa. Dipterans and ephemeropterans were most abundant, followed by plecopterans. Some taxa (e.g. molluscs) were not identified due to a known primer bias and >7,000 OTUs could not be assigned above 85 % similarity. Using the here presented voucher-preserving approach allowed us to identify the pitfalls of DNA metabarcoding as tool for biodiversity assessment in taxonomically unexplored regions such as the Vjosa catchment. However, the comparison of specimen abundance data and molecular data showed the power of non-destructive fixative metabarcoding for detecting MZB communities with highly increased taxonomic resolution.