Fast, reliable, and comprehensive biodiversity monitoring data are needed for environmental decision making and management. Recent work on fish environmental DNA (eDNA) metabarcoding shows that aquatic diversity can be captured fast, reliably, and non-invasively at moderate costs. Because water in a catchment flows to the lowest point in the landscape, often a stream, it can often collect traces of terrestrial species via surface or subsurface runoff along its way or when specimens come into direct contact with water (e.g., for drinking purposes). Thus, fish eDNA metabarcoding data can provide information on fish but also on other vertebrate species that live in riparian habitats. This additional data may offer a much more comprehensive approach for assessing vertebrate diversity at no additional costs. Studies on how the sampling strategy affects species detection especially of stream-associated communities, however, are scarce. We therefore performed an analysis on the effects of biological replication on both fish as well as (semi-)terrestrial species detection. Along a 2 km stretch of the river Mulde (Germany), we collected 18 1-L water samples and analyzed the relation of detected species richness and quantity of biological replicates taken. We detected 58 vertebrate species, of which 25 were fish and lamprey, 18 mammals, and 15 birds, which account for 50%, 24%, and 7% of all native species to the German federal state of Saxony-Anhalt. However, while increasing the number of biological replicates resulted in only 25% more detected fish and lamprey species, mammal, and bird species richness increased disproportionately by 69% and 84%, respectively. Contrary, PCR replicates showed little stochasticity. We thus emphasize to increase the number of biological replicates when the aim is to improve general species detections. This holds especially true, when the focus is on rare aquatic taxa or on (semi-)terrestrial species, the so-called 'bycatch'. As a clear advantage, this information can be obtained without any additional sampling or laboratory effort when the sampling strategy is chosen carefully. With the increased use of eDNA metabarcoding as part of national fish bioassessment and monitoring programs, the complimentary information provided on bycatch can be used for biodiversity monitoring and conservation on a much broader scale.

1. Forest canopies are a highly diverse ecosystem, but despite several decades of intense research, there remain substantial gaps in our knowledge of their biodiversity and ecological interactions. One fundamental challenge in canopy research is the limited accessibility of the ecosystem. Consequently, previous studies have relied on the application of either highly invasive methods such as chemical knockdown, or on time-consuming and expensive setups such as canopy walkways or cranes. Therefore, time- and cost-efficient, ideally minimally invasive yet comprehensive applications are required to help close this knowledge gap. High-throughput metabarcoding of environmental DNA (eDNA) collected from water, soil, or air provides a minimally invasive method for biodiversity assessment, yet its potential for canopy biodiversity monitoring has not been explored. 2. Herein, we conducted metabarcoding of eDNA washed off the canopy via rainwater to explore its monitoring potential. We placed four 1 m2 rain samplers beneath the canopies of four different tree taxa prior to a major rain event, filtered eDNA from the collected rainwater, and performed cytochrome c oxidase subunit I (COI) metabarcoding to profile the invertebrate community. Additionally, we collected and identified all specimens present in the rainwater for verification. 3. We detected 50 invertebrate species by eDNA metabarcoding, of which 43 were not physically present in the water sample, thus likely representing true canopy biodiversity signals. Furthermore, we observed distinct species occurrence patterns corresponding to the four tree taxa, suggesting that ecological patterns such as host specificity can be assessed using the method. 4. In conclusion, our study provides a proof of principle that rainwash eDNA metabarcoding offers a minimally invasive and comprehensive method for tree canopy diversity monitoring.

19The bioluminescent flashlight fish Anomalops katoptron live in schools of several hundred specimens. 20To understand how flashlight fish, integrate bioluminescent signaling into their schooling behavior, 21 we analyzed movement profiles and blink frequencies. Isolated specimen of A. katoptron show a high 22 motivation to align with fixed or moving artificial light organs. Depending on presented frequencies 23 A. katoptron responds with a reduction in swimming speed and its own blink frequency. Higher 24 presented blink frequencies reduce the nearest neighbor distance. In the natural environment 25A. katoptron is changing its blink frequencies and nearest neighbor distance in a context specific 26 manner. Blink frequencies are increased from day to night and during avoidance behavior, while 27 nearest neighbor distance is decreased with increasing blink frequencies. A. katoptron changes its 28 blink frequencies by modifying light organ occlusion. Our results suggest that visually transmitted 29 information via specific blink patterns determine intraspecific communication and group cohesion in 30 schooling A. katoptron. 31 7 determined for 1 Hz and 2 Hz stimulation (1 Hz (0.115 ± 0.008 m/s), 2 Hz (0.119 ± 0.006 m/s), 132 RM ANOVA 0.5 Hz compared to: 1 Hz, p = 0.014; 2 Hz, p = 0.023; Fig. 1D). 133 134