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BACKGROUND: Over the last decade, increasing attention has been directed to using different substrates as sources of environmental DNA (eDNA) in ecological research. Reports on the use of environmental DNA located on the surface of plant leaves and flowers have highlighted the utility of this DNA source in studies including, but not limited to, biodiversity, invasive species, and pollination ecology. The current study assesses grass inflorescence as a source of eDNA for detecting invertebrate taxa. METHODS AND RESULTS: Inflorescences from four common grass species in a central South African grassland were collected for high-throughput sequencing analysis. Universal COI primers were utilised to detect Metazoan diversity. The sequencing results allowed for the detection of three Arthropoda orders, with most OTUs assigned to fungal taxa (Ascomycota and Basidiomycota). Some biases were detected while observing the relative read abundance (RRA) results. DISCUSSION: The observed biases could be explained by the accidental inclusion of invertebrate specimens during sample collection and DNA extraction. Primer biases towards the amplified taxa could be another reason for the observed RRA results. This study provided insight into the invertebrate community associated with the four sampled grass species. It should be noted that with the lack of negative field controls, it is impossible to rule out the influence of airborne eDNA on the observed diversity associated with each grass species. The lack of the inclusion of PCR and extraction blanks in the sequencing step, as well as the inclusion of negative field controls, including other areas for refinement were highlighted, and suggestions were provided to improve the outcomes of future studies.

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While environmental DNA (eDNA) metabarcoding holds promise as a holistic approach to assess vegetation changes and community composition across diverse spatial and temporal scales, systematic investigations of its efficacy compared to conventional field surveys remain scarce in the literature. The present study explores the differences in plant diversity recovered from field surveys and captured with a multi-marker eDNA metabarcoding approach (two nrDNA ITS1 and ITS2, and two cpDNA rbcL and trnL) from river water samples. The eDNA metabarcoding approach retrieved 46 aquatic plants (hydrophytes and helophytes) and 245 terrestrial plants, compared to 24 and 127 species identified from field surveys. On average, eDNA samples collected immediately downstream of the survey sites recovered 43 % and 39 % of the aquatic and terrestrial species observed, respectively. Discrepancies were explained by differences in taxonomic resolution, the stochasticity of the retrieval of rare and elusive species, and the presence of reference sequences. We found a significant positive correlation between spatial and community distances at scales ranging from 2 to 9 km and identified turnover as the driving force of these differences. Metabarcoding demonstrated sensitivity to community changes and both approaches converge on a similar community structure. Interestingly, eDNA samples collected immediately upstream of the survey sites exhibited significant species overlap with the downstream samples (c. 100 m apart). Overall, our results demonstrate that within-site species mismatches between the methods are nonnegligible, and they question the use of eDNA for generating complete species lists at scales comparable to our field surveys (< 100-m transects). However, with adequate sampling and a multi-marker metabarcoding approach, eDNA has the potential to approximate catchment gamma diversity with less sampling effort than conventional surveys.

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Wetlands provide resources, regulate the environment, and stabilize shorelines; however, they are among the most vulnerable ecosystems in the world. The classification of mangrove species allows the determination of the habitat of each species, thereby serving as a basis for determining protection solutions and planning plans for mangrove conservation and restoration according to each environmental condition. We used Phantom 4 multispectral unmanned aerial vehicles (UAVs) to collect data from wetland areas in the Dong Rui Commune, which is one of the most diverse and valuable wetland ecosystems in northern Vietnam. A tree-species classification map was constructed through a combination of the object-based image analysis method and spectral reflectance values of each plant species, and the characteristic distributions of mangrove plants, including Bruguiera gymnorrhiza, Rhizophora stylosa, and Kandelia obovata, were determined with an overall accuracy of 91.11 % and a kappa coefficient (K) of 0.87. The overall accuracy for Rhizophora stylosa was the highest (94.23 %), followed by Kandelia obovata (93.61 %) and Bruguiera gymnorrhiza (85.50 %). An experiment was conducted to map plant taxonomy in the same area based only on a graph of spectral reflectance values at five single-spectral bands, and normalized difference vegetation index values were constructed, resulting in an overall accuracy of 78.22 % and a K of 0.67. The constructed map is useful for classifying, monitoring, and evaluating the structure of each group of mangroves, thereby serving as a basis for determining the distribution of each mangrove species according to natural conditions and contributing to the formulation of policies for afforestation and mangrove conservation in Dong Rui commune.

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Previous difficulties in arthropod taxonomy (such as limitations in conventional morphological approaches, the possibility of cryptic species and a shortage of knowledgeable taxonomists) has been overcome by the powerful tool of DNA barcoding. This study presents a thorough analysis of DNA barcoding in regards to Pakistani arthropods, which were collected from Lahore's Jinnah Garden. The 88 % (9,451) of the 10,792 specimens that were examined were able to generate DNA barcodes and 83% (8,974) of specimens were assigned 1,361 barcode index numbers (BINs). However, the success rate differed significantly between the orders of arthropods, from 77% for Thysanoptera to an astounding 93% for Diptera. Through morphological exams, DNA barcoding, and cross-referencing with the Barcode of Life Data system (BOLD), the Barcode Index Numbers (BINs) were assigned with a high degree of accuracy, both at the order (100%) and family (98%) levels. Though, identifications at the genus (37%) and species (15%) levels showed room for improvement. This underscores the ongoing need for enhancing and expanding the DNA barcode reference library. This study identified 324 genera and 191 species, underscoring the advantages of DNA barcoding over traditional morphological identification methods. Among the 17 arthropod orders identified, Coleoptera, Diptera, Hemiptera, Hymenoptera, and Lepidoptera from the class Insecta dominated, collectively constituting 94% of BINs. Expected malaise trap Arthropod fauna in Jinnah Garden could contain approximately 2,785 BINs according to Preston log-normal species distribution, yet the Chao-1 Index predicts 2,389.74 BINs. The Simpson Index of Diversity (1-D) is 0.989, signaling high species diversity, while the Shannon Index is 5.77, indicating significant species richness and evenness. These results demonstrated that in Pakistani arthropods, DNA barcoding and BOLD are an invaluable tool for improving taxonomic understanding and biodiversity assessment, opening the door for further eDNA and metabarcoding research.

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Environmental DNA (eDNA) analysis has now become a core approach in marine biodiversity research, which typically involves the collection of water or sediment samples. Yet, recently, filter-feeding organisms have received much attention for their potential role as natural eDNA samplers. While the indiscriminate use of living organisms as 'sampling tools' might in some cases raise conservation concerns, there are instances in which highly abundant sessile organisms may become a nuisance as biofouling on artificial marine structures. Here we demonstrate how a sea sponge species that colonizes the moorings of the world's largest curtain of hydroacoustic receivers can become a powerful natural collector of fish biodiversity information. By sequencing eDNA extracted from Vazella pourtalesii retrieved from moorings during routine biofouling maintenance, we detected 23 species of marine fish and mammals, compared to 19 and 15 species revealed by surface and bottom water eDNA respectively, and 28 species captured by groundfish survey in the surrounding area, which are more ecologically impactful and involve higher additional costs. Sponge-based species inventories proved at least as informative as those obtained by traditional survey methods, and are also able to detect seasonal differences in fish assemblages. We conclude that opportunistic sampling of marine sponge biofouling may become an efficient way to document and monitor biodiversity in our rapidly changing oceans.

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Elasmobranch species show low resilience in relation to anthropogenic stressors such as fishing efforts, loss of habitats, and climate change. In this sense, the elasmobranch populations appear to be at risk of extinction in many cases. Despite conservation researchers making efforts to implement knowledge, the information on the biology, reproduction, distribution, or genetic structure of some species is still scattered, often caused by the occurrence of species in inaccessible habitats. Echinorhinus brucus is a deep benthic shark evaluated as "Endangered" on which little information is available, particularly about its geographical range and genetic structure, while E. cookei is listed as "Data Deficient". Echinorhinus brucus belongs to the Echinorhinidae family, and its unique congeneric species is E. cookei. The main morphological diagnostic characteristic of both species is the presence of denticles with different shapes and patterns on the derma. In the present paper, mitochondrial COI and NADH2 sequences were retrieved from both E. brucus and E. cookei species, and analyses were conducted by applying different models of phylogenetic inference. Sequences of E. brucus captured in the Indian Ocean (IOS) did not cluster with the Atlantic E. brucus counterparts (AOS) but instead with E. cookei sequences; the different models showed an overlapping tree topology. Concurrently, a review of the historical and recent captures of the two species was carried out. The worldwide distribution of E. brucus excludes the Pacific Ocean area, where E. cookei occurs, and is characterised by presumably current local extinctions in the North Sea and the western Mediterranean Sea. The dataset describes two definite areas of significantly high abundance of E. brucus located in the Atlantic Ocean (Brazil) and the Indian Ocean (India). These areas suggest zones for conservation plans, especially considering the two lineages identified through molecular approaches.

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Metabarcoding has been widely accepted as a useful tool for biodiversity assessment based on eDNA. The method allows for the detection of entire groups of organisms in a single sample, making it particularly applicable in aquatic habitats. The high sensitivity of the molecular approaches is especially beneficial in detecting elusive and rare fish species, improving biodiversity assessments. Numerous biotic and abiotic factors that affect the persistence and availability of fish DNA in surface waters and therefore affecting species detectability, have been identified. However, little is known about the relationship between the total fish DNA concentration and the detectability of differential abundant species. In this study three controlled mock-community DNA samples (56 individual samples) were analyzed by (i) metabarcoding (MiSeq) of 12S rDNA (175 bp) and by (ii) total freshwater fish DNA quantification (via qPCR of 12S rDNA). We show that the fish DNA quantity affects the relative abundance of species-specific sequences and the detectability of rare species. In particular we found that samples with a concentration between 1000 pg/µL down to 10 pg/µL of total fish DNA revealed a stable relative frequency of DNA sequences obtained for a specific fish species, as well as a low variability between replicates. Additionally, we observed that even in complex mock-community DNA samples, a total fish DNA concentration of 23 pg/µL was sufficient to reliably detect all species in every replicate, including three rare species with proportions of ≤0.5 %. We also found that the DNA barcode similarity between species can affect detectability, if evenness is low. Our data suggest that the total DNA concentration of fish is an important factor to consider when analyzing and interpreting relative sequence abundance data. Therefore, the workflow proposed here will contribute to an ecologically and economically efficient application of metabarcoding in fish biodiversity assessment.

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Monitoring of terrestrial and aquatic species assemblages at large spatial scales based on environmental DNA (eDNA) has the potential to enable evidence-based environmental policymaking. The spatial coverage of eDNA-based studies varies substantially, and the ability of eDNA metabarcoding to capture regional biodiversity remains to be assessed; thus, questions about best practices in the sampling design of entire landscapes remain open. We tested the extent to which eDNA sampling can capture the diversity of a region with highly heterogeneous habitat patches across a wide elevation gradient for five days through multiple hydrological catchments of the Swiss Alps. Using peristaltic pumps, we filtered 60 L of water at five sites per catchment for a total volume of 1800 L. Using an eDNA metabarcoding approach focusing on vertebrates and plants, we detected 86 vertebrate taxa spanning 41 families and 263 plant taxa spanning 79 families across ten catchments. For mammals, fishes, amphibians and plants, the detected taxa covered some of the most common species in the region according to long-term records while including a few more rare taxa. We found marked turnover among samples from distinct elevational classes indicating that the biological signal in alpine rivers remains relatively localised and is not aggregated downstream. Accordingly, species compositions differed between catchments and correlated with catchment-level forest and grassland cover. Biomonitoring schemes based on capturing eDNA across rivers within biologically integrated catchments may pave the way toward a spatially comprehensive estimation of biodiversity.

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Morphological identification of cnidarian species can be difficult throughout all life stages due to the lack of distinct morphological characters. Moreover, in some cnidarian taxa genetic markers are not fully informative, and in these cases combinations of different markers or additional morphological verifications may be required. Proteomic fingerprinting based on MALDI-TOF mass spectra was previously shown to provide reliable species identification in different metazoans including some cnidarian taxa. For the first time, we tested the method across four cnidarian classes (Staurozoa, Scyphozoa, Anthozoa, Hydrozoa) and included different scyphozoan life-history stages (polyp, ephyra, medusa) in our dataset. Our results revealed reliable species identification based on MALDI-TOF mass spectra across all taxa with species-specific clusters for all 23 analysed species. In addition, proteomic fingerprinting was successful for distinguishing developmental stages, still by retaining a species specific signal. Furthermore, we identified the impact of different salinities in different regions (North Sea and Baltic Sea) on proteomic fingerprints to be negligible. In conclusion, the effects of environmental factors and developmental stages on proteomic fingerprints seem to be low in cnidarians. This would allow using reference libraries built up entirely of adult or cultured cnidarian specimens for the identification of their juvenile stages or specimens from different geographic regions in future biodiversity assessment studies.

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In environmental and agronomic settings, even minor imbalances can trigger a range of unpredicted responses. Despite the widespread use of metal-based nanoparticles (NPs) and new bio-nanofertilizers, their impact on crop production is absent in the literature. Therefore, our research is focused on the agronomic effect of spray application of gold nanoparticles anchored to SiO2 mesoporous silica (AuSi-NPs), zinc oxide nanoparticles (ZnO-NPs), and iron oxide nanoparticles (Fe3O4-NPs) on sunflowers under real-world environments. Our findings revealed that the biosynthetically prepared AuSi-NPs and ZnO-NPs were highly effective in enhancing sunflower seasonal physiology, e.g., the value of the NDVI index increased from 0.012 to 0.025 after AuSi-NPs application. The distribution of leaf trichomes improved and the grain yield increased from 2.47 t ha-1 to 3.29 t ha-1 after ZnO-NPs application. AuSi-NPs treatment resulted in a higher content of essential linoleic acid (54.37%) when compared to the NPs-free control (51.57%), which had a higher determined oleic acid. No NPs or residual translocated metals were detected in the fully ripe sunflower seeds, except for slightly higher silica content after the AuSi-NPs treatment. Additionally, AuSi-NPs and NPs-free control showed wide insect biodiversity while ZnO-NPs treatment had the lowest value of phosphorus as anti-nutrient. Contradictory but insignificant effect on physiology, yield, and insect biodiversity was observed in Fe3O4-NPs treatment. Therefore, further studies are needed to fully understand the long-term environmental and agricultural sustainability of NPs applications.

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Monitoring fish communities is central to the evaluation of ecological health of rivers. Both presence/absence of fish species and their relative quantity in local fish assemblages are crucial parameters to measure. Fish communities in lotic systems are traditionally monitored via electrofishing, characterized by a known limited efficiency and high survey costs. Analysis of environmental DNA could serve as a non-destructive alternative for detection and quantification of lotic fish communities, but this approach still requires further insights in practical sampling schemes incorporating transport and dilution of the eDNA particles; optimization of predictive power and quality assurance of the molecular detection method. Via a controlled cage experiment, we aim to extend the knowledge on streamreach of eDNA in small rivers and large brooks, as laid out in the European Water Framework Directive's water typology. Using a high and low source biomass in two river transects of a species-poor river characterized by contrasting river discharge rates, we found strong and significant correlations between the eDNA relative species abundances and the relative biomass per species in the cage community. Despite a decreasing correlation over distance, the underlying community composition remained stable from 25 to 300 m, or up to 1 km downstream of the eDNA source, depending on the river discharge rate. Such decrease in similarity between relative source biomass and the corresponding eDNA-based community profile with increasing distance downstream from the source, might be attributed to variation in species-specific eDNA persistence. Our findings offer crucial insights on eDNA behaviour and characterization of riverine fish communities. We conclude that water sampled from a relatively small river offers an adequate eDNA snapshot of the total fish community in the 300-1000 m upstream transect. The potential application for other river systems is further discussed.

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DNA metabarcoding is routinely used for biodiversity assessment, in particular targeting highly diverse groups for which limited taxonomic expertise is available. Various protocols are currently in use, although standardization is key to its application in large-scale monitoring. DNA metabarcoding of arthropod bulk samples can be conducted either destructively from sample tissue, or nondestructively from sample fixative or lysis buffer. Nondestructive methods are highly desirable for the preservation of sample integrity but have yet to be experimentally evaluated in detail. Here, we compare diversity estimates from 14 size-sorted Malaise trap samples processed consecutively with three nondestructive approaches (one using fixative ethanol and two using lysis buffers) and one destructive approach (using homogenized tissue). Extraction from commercial lysis buffer yielded comparable species richness and high overlap in species composition to the ground tissue extracts. A significantly divergent community was detected from preservative ethanol-based DNA extraction. No consistent trend in species richness was found with increasing incubation time in lysis buffer. These results indicate that nondestructive DNA extraction from incubation in lysis buffer could provide a comparable alternative to destructive approaches with the added advantage of preserving the specimens for postmetabarcoding taxonomic work but at a higher cost per sample.

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Seabirds influence island ecosystems through nutrient additions and physical disturbance. These influences can have opposing effects on an island's invertebrate predator populations. Spiders (order: Araneae) are an important predator in many terrestrial island ecosystems, yet little is known about how seabird presence influences spider communities at the intraisland scale, or how they respond to seasonality in seabird colony attendance.We investigated the effects of seabird presence and seasonality on ground-active spider community structure (activity-density, family-level richness, age class, and sex structure) and composition at the family-level across five short-tailed shearwater breeding islands around south-eastern Tasmania, Australia. Using 75 pitfall traps (15 per island), spiders were collected inside, near, and outside seabird colonies on each island, at five different stages of the short-tailed shearwater breeding cycle over a year. Pitfall traps were deployed for a total of 2674 days, capturing 1592 spiders from 26 families with Linyphiidae and Lycosidae the most common. Spider activity-density was generally greater inside than outside seabird colonies, while family-level richness was generally higher outside seabird colonies. For these islands, seabird breeding stage did not affect activity-densities, but there were some seasonal changes in age class and sex structures with more adult males captured during winter. Our results provide some of the first insights into the spatial and temporal influences seabirds have on spider communities. We also provide some of the first records of spider family occurrences for south-eastern Tasmanian islands, which will provide an important baseline for assessing future change.

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As biodiversity decreases worldwide, the development of effective techniques to track changes in ecological communities becomes an urgent challenge. Together with other emerging methods in ecology, acoustic indices are increasingly being used as novel tools for rapid biodiversity assessment. These indices are based on mathematical formulae that summarise the acoustic features of audio samples, with the aim of extracting meaningful ecological information from soundscapes. However, the application of this automated method has revealed conflicting results across the literature, with conceptual and empirical controversies regarding its primary assumption: a correlation between acoustic and biological diversity. After more than a decade of research, we still lack a statistically informed synthesis of the power of acoustic indices that elucidates whether they effectively function as proxies for biological diversity. Here, we reviewed studies testing the relationship between diversity metrics (species abundance, species richness, species diversity, abundance of sounds, and diversity of sounds) and the 11 most commonly used acoustic indices. From 34 studies, we extracted 364 effect sizes that quantified the magnitude of the direct link between acoustic and biological estimates and conducted a meta-analysis. Overall, acoustic indices had a moderate positive relationship with the diversity metrics (r = 0.33, CI [0.23, 0.43]), and showed an inconsistent performance, with highly variable effect sizes both within and among studies. Over time, studies have been increasingly disregarding the validation of the acoustic estimates and those examining this link have been progressively reporting smaller effect sizes. Some of the studied indices [acoustic entropy index (H), normalised difference soundscape index (NDSI), and acoustic complexity index (ACI)] performed better in retrieving biological information, with abundance of sounds (number of sounds from identified or unidentified species) being the best estimated diversity facet of local communities. We found no effect of the type of monitored environment (terrestrial versus aquatic) and the procedure for extracting biological information (acoustic versus non-acoustic) on the performance of acoustic indices, suggesting certain potential to generalise their application across research contexts. We also identified common statistical issues and knowledge gaps that remain to be addressed in future research, such as a high rate of pseudoreplication and multiple unexplored combinations of metrics, taxa, and regions. Our findings confirm the limitations of acoustic indices to efficiently quantify alpha biodiversity and highlight that caution is necessary when using them as surrogates of diversity metrics, especially if employed as single predictors. Although these tools are able partially to capture changes in diversity metrics, endorsing to some extent the rationale behind acoustic indices and suggesting them as promising bases for future developments, they are far from being direct proxies for biodiversity. To guide more efficient use and future research, we review their principal theoretical and practical shortcomings, as well as prospects and challenges of acoustic indices in biodiversity assessment. Altogether, we provide the first comprehensive and statistically based overview on the relation between acoustic indices and biodiversity and pave the way for a more standardised and informed application for biodiversity monitoring.

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The analysis of environmental DNA (eDNA) is revolutionizing the monitoring of biodiversity as it allows to assess organismic diversity at large scale and unprecedented taxonomic detail. However, eDNA consists of an extracellular and intracellular fraction, each characterized by particular properties that determine the retrievable information on when and where organisms live or have been living. Here, we review the fractions of eDNA, describe how to obtain them from environmental samples and present a four-scenario concept that aims at enhancing spatial and temporal resolution of eDNA-based monitoring. Importantly, we highlight how the appropriate choice of eDNA fractions precludes misinterpretation of eDNA-based biodiversity data. Finally, future avenues of research towards eDNA fraction-specific analyses are outlined to unravel the full potential of eDNA-based studies targeting micro- and macro-organisms.

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Nature-Based Solutions (NBS) can be defined as solutions based on natural processes that meet societal challenges and simultaneously provide human well-being and biodiversity benefits. These solutions are envisioned to contribute to operationalizing sustainable development strategies, especially in the context of adaptation to climate change (e.g. flood risk reduction). In order to quantify NBS performance, ease their uptake and advocate for them as alternatives to "business-as-usual" infrastructures, a comprehensive, holistic valuation of their multiple benefits (multiple advantages and disadvantages) is needed. This entails quantifying non-market benefits for people and nature in addition to determining the (direct) cost-benefit of the risk-reduction measure. Despite the importance given to the assessment of non-tangible benefits for people and nature in the literature, systematic data collection on these dimensions seems to be missing. This study reviews publications that used stated preference methods to assess non-market human benefits of NBS and NBS-like strategies. Its aim is to highlight any biases or knowledge gaps in this kind of evaluation. Our results show that the valuation of non-tangible benefits of NBS (e.g. increased recreation and well-being, enhanced biodiversity) still suffers from a lack of common framing. Despite some steps being taken on enabling interconnected benefit assessments, unexploited opportunities concerning the integrated assessment of non-market human and nature benefits predominate. Moreover, the research to-date appears based on a case-to-case approach, and thus a shared holistic method does not emerge from the present literature, potentially delaying the uptake of NBS. We argue that future research could minimize missed opportunities by focusing on and systematically applying holistic benefits assessments. Methods based on stated preference surveys may help to ensure holistic approaches are taken, as well as contributing to their replicability and application when upscaling NBS.

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The interface between field biology and technology is energizing the collection of vast quantities of environmental data. Passive acoustic monitoring, the use of unattended recording devices to capture environmental sound, is an example where technological advances have facilitated an influx of data that routinely exceeds the capacity for analysis. Computational advances, particularly the integration of machine learning approaches, will support data extraction efforts. However, the analysis and interpretation of these data will require parallel growth in conceptual and technical approaches for data analysis. Here, we use a large hand-annotated dataset to showcase analysis approaches that will become increasingly useful as datasets grow and data extraction can be partially automated.We propose and demonstrate seven technical approaches for analyzing bioacoustic data. These include the following: (1) generating species lists and descriptions of vocal variation, (2) assessing how abiotic factors (e.g., rain and wind) impact vocalization rates, (3) testing for differences in community vocalization activity across sites and habitat types, (4) quantifying the phenology of vocal activity, (5) testing for spatiotemporal correlations in vocalizations within species, (6) among species, and (7) using rarefaction analysis to quantify diversity and optimize bioacoustic sampling.To demonstrate these approaches, we sampled in 2016 and 2018 and used hand annotations of 129,866 bird vocalizations from two forests in New Hampshire, USA, including sites in the Hubbard Brook Experiment Forest where bioacoustic data could be integrated with more than 50 years of observer-based avian studies. Acoustic monitoring revealed differences in community patterns in vocalization activity between forests of different ages, as well as between nearby similar watersheds. Of numerous environmental variables that were evaluated, background noise was most clearly related to vocalization rates. The songbird community included one cluster of species where vocalization rates declined as ambient noise increased and another cluster where vocalization rates declined over the nesting season. In some common species, the number of vocalizations produced per day was correlated at scales of up to 15 km. Rarefaction analyses showed that adding sampling sites increased species detections more than adding sampling days.Although our analyses used hand-annotated data, the methods will extend readily to large-scale automated detection of vocalization events. Such data are likely to become increasingly available as autonomous recording units become more advanced, affordable, and power efficient. Passive acoustic monitoring with human or automated identification at the species level offers growing potential to complement observer-based studies of avian ecology.

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Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to profile biota in basic and applied biodiversity research because of its targeted nature that allows sequencing of genetic markers from many samples in parallel. To achieve this, PCR amplification is carried out with primers designed to target a taxonomically informative marker within a taxonomic group, and sample-specific nucleotide identifiers are added to the amplicons prior to sequencing. The latter enables assignment of the sequences back to the samples they originated from. Nucleotide identifiers can be added during the metabarcoding PCR and during "library preparation", that is, when amplicons are prepared for sequencing. Different strategies to achieve this labelling exist. All have advantages, challenges and limitations, some of which can lead to misleading results, and in the worst case compromise the fidelity of the metabarcoding data. Given the range of questions addressed using metabarcoding, ensuring that data generation is robust and fit for the chosen purpose is critically important for practitioners seeking to employ metabarcoding for biodiversity assessments. Here, we present an overview of the three main workflows for sample-specific labelling and library preparation in metabarcoding studies on Illumina sequencing platforms; one-step PCR, two-step PCR, and tagged PCR. Further, we distill the key considerations for researchers seeking to select an appropriate metabarcoding strategy for their specific study. Ultimately, by gaining insights into the consequences of different metabarcoding workflows, we hope to further consolidate the power of metabarcoding as a tool to assess biodiversity across a range of applications.

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Two species of Anacaena Thomson, 1859, A.angatbuhay sp. nov. and A.auxilium sp. nov., are described from Northern Luzon, Philippines. The new species can be distinguished through colour, body shape, surface puncturation and characteristic aedeagi. Descriptions are provided and complemented with habitus photographs and drawings of the aedeagi. Data on genus distribution in the Philippines are reviewed and an updated Philippine checklist is provided.

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Two new species of Limnichidae beetles, Byrrhinusnegrosensis sp. nov. and Byrrhinusvillarini sp. nov., are described from the Island of Negros in the Philippines. The adult specimens of the new species can be differentiated by patterns of body punctation, colour and orientation of elytral pubescence, posterolateral angle of pronotum, tarsomere length ratio and aedeagal form. Two clades, representing the two new species, were retrieved in the Maximum Likelihood gene tree using the 3'-end of the COI gene. Maximum genetic divergence within B.negrosensis sp. nov. and B.villarini sp. nov. were recorded to be 2.3% and 1.3%, respectively, while the mean interspecific divergence between the two new species was 19.7%. Morphological descriptions, digital photographs and COI sequences were provided for the two species. The state of knowledge of Byrrhinus is reviewed and an updated Philippine checklist is provided. By coupling morphological and molecular data, this paper provides the first additional new species of Philippine Byrrhinus in the last 28 years.