Environmental DNA

Freshwater biomonitoring is essential for aquatic biodiversity conservation. Advances in high-throughput sequencing allowed parallel sequencing of community samples containing DNA from environmental samples, i.e., metabarcoding. Two approaches of DNA-based method are widely used, bulk-sample metabarcoding the use of bulk tissues such as insects and eDNA the use of environmental samples such as air, water and soil. Despite the novelty of this approach for routine freshwater biomonitoring, questions still need to be answered about its applicability and reliability due to confounding factors, e.g., sample type, laboratory technicalities, and limitations of databases. Hence, studies on direct comparisons are essential to validate the efficiency of these molecular approaches compared to the conventional morphological approach to accurately assessed biodiversity for riverine benthic macroinvertebrate biomonitoring. This study used three approaches to estimate diversity and composition of benthic macroinvertebrates. We also evaluated the relationship between benthic macroinvertebrate communities and environmental factors. We morphologically identified 8,052 individuals from 35 families, 31 genera, and 29 species. eDNA metabarcoding identified 51 families, 84 genera, and 90 species, while 37 families, 55 genera, and 107 species were detected through bulk-sample metabarcoding. We report that bulk-sample metabarcoding showed finer taxonomic resolution than other approaches. Our study highlights the use of bulk-sample metabarcoding for macroinvertebrate biodiversity assessment.

Airborne environmental DNA (eDNA) has shown promise as a terrestrial biomonitoring tool and its ecological applications are expanding. Despite its growing use, airborne eDNA does not yet have the extensive body of supporting research like its aquatic counterpart, with considerable uncertainty remaining concerning how airborne eDNA behaves, with regards to signal duration, and how much sampling effort is needed to capture DNA in a given airspace. By using airborne eDNA in a semi-controlled environment which acted as an artificial roost where bat species and their abundances were known, we estimated the sampling intensity (both the number of samples and number of sampling events) required to capture bat diversity of a given airspace, as well as signal persistence of airborne eDNA. Together these data provide a temporal scale for airborne eDNA measurements. The majority of species richness was detected using as little as 4 samplers in this enclosed space and the greater the number of sampling events, the fewer samplers were needed. Both air movement and the type of environment (i.e., enclosed space, open area etc.) are likely to impact detection and need to be considered during study design. eDNA also appeared to settle out of the air quickly, suggesting that detections likely reflect recent activity, which also has important implication for rare species which may only have a narrow window for detection. Our results add to the growing body of literature that indicate airborne eDNA can be a useful biosurvey method, especially for rapid surveys in communities with high turnover rates.

Early detection of invasive alien species is essential for preventing establishment and mitigating ecological impacts, particularly in island ecosystems harbouring evolutionarily isolated endemic species. Recently, despite increasing reptile introductions and their suggested widespread impacts, methods for monitoring arboreal invasive reptiles remain limited. This study addressed the need for practical detection tools by developing terrestrial environmental DNA (eDNA) sampling methods that collect DNA from leaf surfaces to detect the arboreal invasive green anole (Anolis carolinensis) in the Ogasawara Islands, Japan. Two methods, wiping leaf surfaces with gauze and rinsing with sprayed water, were tested. At an invaded site, green anoles were successfully detected in eight out of 10 samples in both methods. Although no significant difference in eDNA concentration was observed, the wiping method was selected for its greater simplicity. Subsequently, the relationship between green anole population density and eDNA concentrations detected using the wiping method was investigated, suggesting a significant positive relationship. This is the first report demonstrating that terrestrial eDNA concentrations can reflect arboreal reptile population density, suggesting potential applications in quantitative terrestrial biodiversity assessments. Furthermore, the successful detection of eDNA even in the extremely low-density habitat of the green anole demonstrates the usefulness of eDNA-based surveys for early detection of invasions. The method developed here may be broadly applicable to terrestrial biodiversity monitoring, especially in tree-dwelling taxa. Given accelerating biological invasions and biodiversity loss, this approach is expected to benefit managers, conservationists, and researchers concerned with terrestrial ecosystems.

DNA based methods offer a rapid and cost-effective way for detecting species occurrence and monitoring biodiversity; among them bulk DNA metabarcoding is well-established, and recently developed environmental DNA (eDNA)-based methods offer a non-destructive alternative. With a goal to develop suitable methods for assessing insect biodiversity in ecosystems for which DNA reference libraries are not well developed and incomplete, such as remote islands, we compared established bulk DNA metabarcoding methods with eDNA across three replicated terrestrial ecosystem types (limestone forest, degraded forest, and grassland) in Guam. Using two mitochondrial COI primer pairs, we performed bulk DNA metabarcoding of standard entomological collection methods (malaise traps, pan traps, vegetation beating), and compared the assessment of biodiversity with that from different eDNA sources (flowers, spider webs, leaves, tree trunks). In our samples, eDNA and bulk DNA metabarcoding both detected a large proportion of overall taxa (OTUs, 86.6% and 60.3%, respectively). Although DNA metabarcoding detected significantly more taxa, eDNA proved to be a reasonable non-destructive alternative. As expected, because of limitations in existing reference databases for remote habitats, species-level identification was achieved for only a few OTUs. Overall, the sampling approach was the dominant driver of arthropod diversity, explaining [~]17% of the observed variation, while habitat type accounted for [~]4%. Thus, each sampling approach captured some unique diversity signals and contributed to the complementary effect of maximizing detection. For rapid insect biodiversity surveys of terrestrial arthropods, we recommend an integrated metabarcoding approach, and in sensitive habitats where insect capture is undesirable, eDNA offers a powerful alternative to monitor diversity and community change.

O_LIInformation on alpha (local), beta (between habitats), and gamma (regional) diversity is fundamental to understanding biodiversity as well as the function and stability of community dynamics. Methods like environmental DNA (eDNA) metabarcoding are currently considered useful to investigate biodiversity. C_LIO_LIWe compared the performance of eDNA metabarcoding with visual and capture surveys for estimating alpha and gamma diversity of river fish communities, and nestedness and turnover in particular. C_LIO_LIIn five rivers across west Japan, by comparison to visual/capture surveys, eDNA metabarcoding detected more species in the study sites (i.e., alpha diversity). Consequently the overall number of species in the region (i.e., gamma diversity) was higher. In particular, the species found by visual/capture surveys were encompassed by those detected by eDNA metabarcoding. C_LIO_LIEstimates of community diversity within rivers differed between survey methods. Although we found that the methods show similar levels of community nestedness and turnover within the rivers, visual/capture surveys showed more distinct community differences from upstream to downstream. Our results suggest that eDNA metabarcoding may be a suitable method for community assemblage analysis, especially for understanding regional community patterns, for fish monitoring in rivers. C_LI

The study of insect decline remains a major frontier in insect biodiversity and conservation. Despite growing concern about accelerating rates of insect decline generally, relatively little data has been compiled about species of aquatic insects. Data is particularly lacking on the distribution of aquatic insects in urban ecosystems. Here, we compare environmental DNA (eDNA) metabarcoding and community science observation as means of monitoring Odonata within an urban system in Southwest Idaho. We show that the distribution of Odonata across this urban landscape is not uniform and that both monitoring methods have different strengths and weaknesses. We found that eDNA metabarcoding was very sensitive to the identification of genera from underrepresented families in the region, but was unable to distinguish between closely related genera, particularly from localities where eDNA could accumulate more damage. On the other hand, community science observations effectively identified the presence of genera from more speciose families but missed the presence of relatively rare species, and those that had a short flight season. These findings suggest that, in our study system, eDNA and community science are highly complementary of each other. In cases where only one method is employed for a monitoring or conservation project, care should be given to account for the biases of each approach.

Environmental DNA (eDNA) analysis is a cost-effective and noninvasive tool for species and biodiversity monitoring in aquatic environments. Passive eDNA sampling is a novel alternative to conventional sampling methods such as water filtration. In this study, we examined the efficacy of the sponge skeleton as a passive eDNA sampler for monitoring freshwater fish. The performance of the passive sampling method was compared with that of standard water filtration in a river environment. Five DNA extraction methods were used in three experiments, and a suitable method for DNA extraction from sponge skeleton was identified. Quantitative fish metabarcoding using MiFish primers revealed no significant differences in species richness between the aqueous and passive sampling methods. Although both the sampling methods showed comparable trends in fish community structure, different clusters were identified for water sampling and passive samplers based on the differences in the DNA concentration of each fish species. The fish diversity in the passive samples was comparable among four DNA extraction methods (except for the filtration method) using the direct capture method. Our results demonstrate the efficacy of passive eDNA sampling for monitoring freshwater fish diversity and the potential use of sponge skeletons as absorption materials for passive eDNA samplers.

Environmental DNA (eDNA) has become an established and efficient method for monitoring biodiversity in aquatic systems. However, there is a need to compare and standardise sampling methods across ecosystem types, particularly complex ecosystems such as estuaries where unique challenges for monitoring fish populations are present due to fluctuating environmental factors. Here, we compare fish biodiversity metrics obtained from eDNA metabarcoding data using four different eDNA filtering methods: three manual filtering methods with different pore sizes (0.45, 1.2 and 5 {micro}m) and a newly established passive method, the metaprobe. The study was applied across a salinity gradient in a hyper-tidal estuarine ecosystem. Overall, 44 fish species were detected across the four methods used. The 0.45 {micro}m filter recovered the highest richness (39 species), then the metaprobe method (35), followed by the 1.2 {micro}m (34) and 5 {micro}m (33) filters. Filter performance between salinity gradients revealed that the 0.45 {micro}m and the 1.2 {micro}m methods recovered the highest species richness across all sampled zones. The 0.45 {micro}m also had the most consistent detection probabilities using representative species from each zone. While the 0.45 {micro}m method appeared to be the optimal method, each of the methods can be considered as a viable and comparable option for biomonitoring in dynamic ecosystems such as estuaries and rivers. In particular, the passive metaprobe (used in a freshwater system for the first time here) performed well in comparison to the manual filtering methods despite a short deployment time. This study provides critical insights for optimising fish biodiversity assessments using eDNA metabarcoding in estuarine ecosystems, providing a valuable framework for future monitoring efforts in similar systems worldwide.

Environmental DNA (eDNA) provides a powerful tool for biodiversity monitoring in large river ecosystems. However, current studies often rely on subjective site selection and lack systematic sampling designs. This can limit the ability to capture the full spectrum of environmental conditions that species depend on, thereby compromising sampling efficiency. To address this challenge, we propose utilizing remote sensing-based clustering for environmental stratification of sampling designs, thereby enhancing detection capabilities and increasing the objectivity of eDNA sampling. Using GBIF-based fish species distribution models and simulated distributions along the Danube, we demonstrate that this approach enhances detection efficiency compared to conventional random and regular sampling methods. To facilitate practical implementation, we developed a tool to help fieldwork planners of river sampling campaigns automatically apply this method and select stratified sampling sites without the need for extensive data processing. Finally, we demonstrate that eDNA detection occurred most frequently within the range of 0-20km downstream of the expected modeled distribution of species, suggesting that the diffusion of the signal should be further considered in the sampling design process. Our findings highlight the potential of remote sensing-based stratification to create more efficient and objective sampling designs but suggest that sampling design should be further combined with hydrological information to optimize cost-efficient sampling. The development of standard and robust sampling protocols will help advance more cost-effective eDNA-based biodiversity monitoring in riverine ecosystems.

Declines in population and diversity of North American bats are rapidly occurring due to habitat loss, incidental take from various industry projects, and lethal White-nose Syndrome disease. It is critical to accurately census habitat for appropriate conservation measures, yet traditional sampling methodology, such as mist netting and acoustic recordings, can be time-intensive and biased. Instead, a passive sampling tool that does not rely on the a priori knowledge of bat roosts may provide crucial information on bat communities. In the water-limited habitats of forested uplands of the Appalachian Plateau, water-filled road-ruts are important resources for bats. Therefore, we developed an environmental DNA (eDNA) protocol to sample isolated road-ruts that may have the presence of sloughed cellular material from actively drinking bats. The detection of bat eDNA was investigated from a positive control experiment, and across 47 water samples collected in Kentucky and Ohio. Water samples were analyzed using both species-specific quantitative polymerase chain reaction (qPCR) and community metabarcoding methodologies. Using qPCR analysis, we detected eDNA from big brown bat (Eptesicus fuscus) and eastern red bat (Lasiurus borealis) from water-filled road-ruts. While the community metabarcoding approach failed to detect any bat eDNA, many non-target amphibians, birds, and mammals were identified. These results suggest eDNA found within road-ruts provides an additional detection tool for surveying biodiversity across upland forests. Additionally, the use of qPCR increased the detection of rare eDNA targets, which will be crucial for properly implementing future eDNA applications for improving bat conservation efforts across the landscape. Article impact statementEnvironmental DNA provides detection of bats from drinking sources offering a novel survey method for management and conservation efforts

Environmental DNA (eDNA) exists in three states: membrane-bound, adsorbed, and dissolved. These states differ in persistence and degradation, strongly influencing the interpretation of eDNA data. Despite this, they have rarely been separated and analyzed independently from environmental samples. We developed a state-sorting workflow to isolate and analyze them, applying it to samples from 221 sites from 58 streams across eight lake watersheds with COI and ITS metabarcoding to reveal differences in biodiversity content and transport dynamics. Our results show that all three states contain both shared and unique taxonomic diversity for COI, and that plant DNA was only detected in the membrane-bound state. For COI, membrane-bound eDNA contained 87.8% of observed ASVs, far exceeding the adsorbed (37.2%) and dissolved (20.5%) states. Only membrane-bound eDNA showed evidence of downstream transport, but its extent varied among watersheds due to local hydrology. While upstream eDNA was transported to stream-lake confluences, lake surface samples showed marked turnover in community composition. Clarifying the fate of membrane-bound eDNA within lakes will enhance catchment-level detection from lake samples and understanding of lake hydrodynamics. Of the environmental parameters assessed, water temperature was most strongly aligned with changes in community composition between sites. Most previous studies have likely captured the majority of the eDNA diversity within their samples by inadvertently targeting the membrane-bound state. This study demonstrates the utility of eDNA state-sorting, but the methods require further refinement. Analyzing states independently improves the interpretation of eDNA data and elucidates the processes governing eDNA fate and transport.

Environmental DNA (eDNA) analysis can detect aquatic organisms, including rare and endangered species, in a variety of habitats. The degradation of eDNA concentration is important to investigate their distribution and has also been experimentally evaluated. It is important to integrate these data to synthesize eDNA degradation in various environments. We collected the eDNA degradation rates and related factors, especially water temperature and fragment lengths of the measured DNA from 28 studies. Our results suggest that water temperature and fragment length are significantly related to the eDNA degradation rate. From the 95% quantile model simulation, we predicted the maximum eDNA degradation rate in various combinations of water temperature and fragment length. Predicting eDNA degradation could be important for evaluating species distribution and inducing innovation of eDNA methods, especially for rare and endangered species with lower DNA concentrations.

DNA-based approaches utilizing high-throughput sequencing (HTS) (e.g. DNA metabarcoding) have revolutionized ecological biomonitoring by providing higher sample throughput, greater reproducibility, and better cost-benefits compared to traditional morphology-based bioassessment studies. Here, we utilized DNA metabarcoding in a watershed in Ontario (Canada) dominated by agricultural land uses. Our aim is to understand patterns of biodiversity in benthic taxa from data generated and inferred at various taxonomic scales and to compare these findings with over a decade of traditional morphological data. We sampled 18 watercourses during summer and fall 2023, spanning a forested-to-agricultural land-use gradient. We found significant differences between metabarcoding and historical morphology data where DNA provided more richness values at both the species (p = 2x10-5) and order (p = 0.008) levels. Whereas the morphology dataset contained many unresolved taxa, DNA metabarcoding captured a broader taxonomic breadth with diverse genetic profiles among taxa. Non-metric multidimensional scaling (NMDS) analyses on DNA metabarcoding data produced tighter clusters, more precise separation by land use, and greater consistency across taxonomic scales. Both urban context and land use had significant associations with metabarcoding patterns observed, with differences being strongest between agriculturally-dominated and primarily forested sites (median R{superscript 2} {approx} 0.08-0.11). We also found strong, consistent environmental signals linked to agricultural settings, such as water conductivity and turbidity, and pH. Altogether, our DNA-based results demonstrate the differences in community composition among different land uses in this watershed. Importantly, our work highlights the need for more taxonomic resolution (obtained through DNA analysis) to decipher community changes linked to anthropogenic and environmental drivers, as morphological data alone may lack the precision needed to capture these patterns.

Freshwater ecosystems, such as streams, are facing increasing pressures from agricultural land use. Aquatic insects and other macroinvertebrates have historically been used as indicators of ecological condition and water quality in freshwater biomonitoring programs; however, many of these protocols use coarse taxonomic resolution (e.g., family) when identifying macroinvertebrates. The use of family-level identification can mask species-level diversity, as well as patterns in community composition in response to environmental variables. Recent literature stresses the importance of robust biomonitoring to detect trends in insect decline globally, though most of these studies are carried out in terrestrial habitats. Here, we incorporate molecular identification (DNA metabarcoding) into a stream biomonitoring sampling design to explore the diversity and variability of aquatic macroinvertebrate communities at small spatial scales. We sampled twenty southern Ontario streams in an agricultural landscape for aquatic macroinvertebrates and, using DNA metabarcoding, revealed incredibly rich benthic communities which were largely comprised of rare taxa detected only once per stream despite multiple biological replicates. In addition to numerous rare taxa, our species pool estimates indicated that after 240 samples from twenty streams, there was a large proportion of taxa present which remained undetected by our sampling regime. When comparing different levels of taxonomic resolution, we observed that using OTUs revealed over ten times more taxa than family-level identification. A single insect family, the Chironomidae, contained over one third of the total number of OTUs detected in our study. Within-stream dissimilarity estimates were consistently high for all taxonomic groups (invertebrate families, invertebrate OTUs, chironomid OTUs), indicating stream communities are very dissimilar at small spatial scales. While we predicted that increased land use would homogenize benthic communities, this was not supported as within-stream dissimilarity was unrelated to land use.

Vertebrates play vital roles in maintaining ecosystem processes and services and serve as valuable indicators of environmental health, making them an important target for monitoring and conservation efforts. Within the environmental DNA (eDNA) toolbox, airborne environmental DNA has recently emerged as a novel approach for vertebrate monitoring. In this study, we evaluated on-site airborne eDNA for terrestrial vertebrate monitoring in the Luangwa Valley savanna in Zambia, which represents a major biodiversity stronghold of largely intact wilderness and with high levels of vertebrate diversity and endemism. Six air samplers were deployed over four days alongside camera traps for validation, and samples were processed using a mobile molecular laboratory. In total, 120 terrestrial vertebrate taxa were detected with airborne eDNA, including 16 of the 17 taxa recorded by camera traps, demonstrating high sensitivity. Notably, 72.5% of taxa were detected on the first day, and a single sampler recovered 61.7% of all taxa; the taxonomic richness incrementally increased with extended sampling efforts, but the magnitude of these increases declined progressively. The detected taxa spanned the four terrestrial vertebrate classes and encompassed a wide range of ecological traits. These results show that airborne eDNA can quickly recover a substantial and representative fraction of local vertebrate diversity within a short sampling window, while extended sampling can improve detection of less common taxa. Despite existing limitations, our findings support the use of airborne eDNA as an efficient and scalable complementary tool for community-level biodiversity assessments in terrestrial ecosystems such as Zambezian savannas.

Environmental DNA (eDNA) methods offer a powerful tool for monitoring aquatic species, yet field applications remain challenged by uncertainty in DNA transport, mixing, and detection, particularly in flowing or tidally influenced systems. One approach to improve confidence in eDNA surveys is the use of controlled DNA sources (positive controls), but questions remain regarding how the biological condition of the source influences eDNA release and detectability. This study evaluated differences in eDNA concentrations emitted from live versus dead fish in a controlled, shallow, well-mixed channel. Using a fixed point-sampling design, we measured eDNA concentrations over time and modeled the effects of treatment, sampling time, temperature, and water velocity. Dead fish consistently released significantly higher concentrations of eDNA than live fish, while eDNA concentrations declined over time in both treatments. Water temperature and velocity did not significantly influence detection, and the rate of eDNA decline was similar between live and dead treatments. These findings highlight the importance of source condition and site-specific mixing dynamics when interpreting positive control experiments and underscore the value of site characterization when designing eDNA sampling protocols.

Biodiversity is in decline due to human land use, exploitation, and climate change. To be able to counteract this alarming trend it is paramount to closely monitor biodiversity at global scales. Because this is practically impossible with traditional methods, the last decade has seen a strong push for solutions. In aquatic ecosystems the monitoring of species from environmental DNA (eDNA) has emerged as one of the most powerful tools at our disposal but in terrestrial ecosystems the power of eDNA for monitoring has so far been hampered by the local scale of the samples. In this study we report the first attempt to detect insects from airborne eDNA. We compare our results to two traditional insect monitoring projects (1) using light trapping for moth monitoring and (2) transect counts for the monitoring of butterflies and wild bees. While we failed to detect many of the same species monitored with the traditional methods, airborne eDNA metabarcoding revealed DNA from from six classes of Arthropods, and twelve order of Insects - including representatives from all of the four largest orders: Diptera (flies), Lepidoptera (butterflies and moths), Coleoptera (beetles) and Hymenoptera (bees, wasps and ants). We also recovered DNA from nine species of vertebrates, including frogs, birds and mammals as well as from 12 other phyla. We suggest that airborne eDNA has the potential to become a powerful tool for terrestrial biodiversity monitoring, with many impactful applications including the monitoring of pests, invasive or endangered species or disease vectors.

Unionid freshwater mussels exhibit a unique form of mitochondrial inheritance, termed doubly uniparental inheritance, in which a maternal and a paternal mitotype is transmitted uniparentally. The exclusive presence of a male mitotype in gonadal tissue and sperm cells suggests that environmental DNA (eDNA) could serve as a non-invasive method for monitoring freshwater mussel reproduction. Yet, the dynamics of male mitotype detection within the environment remain poorly understood. This study analyzed seasonal eDNA samples from two diverse mussel beds, detecting 24 mitochondrial operational taxonomic units (MOTUs) associated with the male mitotype. Peaks in male mitotype signal for mussels identifiable to the species level generally aligned with expected spawning periods based on female gravidity records (e.g., Pyganodon grandis, Lasmigona costata, Ortmaniana ligamentina). Additionally, male mitotype detection was often sporadic compared to the consistently detected female mitotype, indicating that male signals may be tied to behavioral or reproductive events rather than continuous shedding. While elevated male signals may reflect spawning, alternative sources such as tissue decay, mitotype leakage, glochidia release, or post-spawning gamete clearance complicate interpretation. A male-to-female mitotype ratio is proposed as a more reliable proxy for identifying sperm release events, given the high concentration of male mitotypes that occurs within spermatozeugmata. Limitations in male mitotype reference databases hindered species-level resolution for many MOTUs, underscoring the need for expanded genomic resources. Overall, this work demonstrates that male mitotype eDNA likely provides valuable insights into mussel reproductive ecology, while emphasizing the importance of long-term monitoring and integrated gametogenesis studies to refine its application in conservation.

Environmental DNA (eDNA) provides a powerful non-invasive tool for monitoring freshwater mussel assemblages, yet detection probabilities can be influenced by reproductive behaviors, seasonal vertical migration, and hydrological conditions. This study assessed eDNA detection from April through October across two diverse mussel beds in Ohio, encompassing species with both tachytictic (short-term brooders) and bradytictic (long-term brooders) reproductive strategies. Mussel DNA was consistently detected across seasons, with detection patterns generally aligning with species observed through a visual tactile survey. Overall, the eDNA sequence abundance was positively correlated with tactile mussel counts, however congruence between the two surveys was strongest during low discharge and when the surveys occurred in close temporal proximity to one another. This study finds that eDNA sampling for freshwater mussels performs adequately within the currently prescribed survey window for visual surveys. However, seasonal factors such as endobenthic burial behavior and high discharge events may have reduced detection efficiency, particularly in Killbuck Creek, where species richness was lowest during periods of high flow in early spring. Therefore, decisions made regarding the timing of eDNA surveys should consider local environmental conditions (e.g., temperature and flow) to achieve optimal results.

Bioassessment of freshwater quality via eDNA is rapidly developing into a powerful alternative to traditional methods involving collecting, sorting, and identifying macroinvertebrates based on morphology. Particularly attractive would be methods that can use remote-controlled boats for sampling because it would allow for cost-effective, and frequent monitoring at multiple sites. The latter will be particularly important for tropical reservoirs that require year-around surveillance. We here optimize molecular protocols for capturing reservoir-specific differences in metazoan communities based on small water volumes (15 mL). The optimization is based on samples from two freshwater reservoirs with very different water qualities ("reservoir signal"). Each reservoir was sampled at three sites ("biological replicates"). For each water sample, the DNA was extracted twice ("technical replicates"). We then tested how much DNA template (0.1 ng to 15 ng) and how many PCR cycles (25 or 35) minimized variance between technical replicates. We find that 15 mL is sufficient for capturing the reservoir signal regardless of sampling time, template amounts, or PCR cycle numbers. Indeed, extrapolation from our results suggests that <1 mL would be sufficient because only 17 of 59 metazoan mOTUs (mainly planktonic crustaceans and rotifers) detected with a 313bp COI minibarcode were shared. We find that the use of 35 PCR cycles significantly lowered the number of detected species and that template amounts <0.5 ng yielded somewhat higher variance between technical replicates. Despite extensive trials, the variance between technical replicates remained high (Bray-Curtis: 5-20%; Jaccard: 10-40%) and we predict that it will be difficult to reduce this variance further. However, the overall reservoir differences are so strong that all biological and technical replicates can be correctly assigned.