ABSTRACT1. Detecting aquatic macroorganisms with environmental DNA (eDNA) is a new survey method with broad applicability. However, the origin, state, and fate of aqueous macrobial eDNAwhich collectively determine how well eDNA can serve as a proxy for directly observing organisms and how eDNA should be captured, purified, and assayed -are poorly understood. 2.The size of aquatic particles provides clues about their origin, state, and fate. We used sequential filtration size fractionation to measure, for the first time, the particle size distribution (PSD) of macrobial eDNA, specifically Common Carp (hereafter referred to as Carp) eDNA. We compared it to the PSDs of total eDNA (from all organisms) and suspended particle matter (SPM). We quantified Carp mitochondrial eDNA using a custom qPCR assay, total eDNA with fluorometry, and SPM with gravimetric analysis. 3.In a lake and a pond, we found Carp eDNA in particles from >180 to <0.2 !m, but it was most abundant from 1-10 !m. Total eDNA was most abundant below 0.2 !m and SPM was most abundant above 100 !m. SPM was "0.1% total eDNA, and total eDNA was "0.0004% Carp eDNA. 0.2 !m filtration maximized Carp eDNA capture (85%±6%) while minimizing total (i.e., non-target) eDNA capture (48%±3%), but filter clogging limited this pore size to a volume <250 mL. To mitigate this limitation we estimated a continuous PSD model for Carp eDNA and derived an equation for calculating isoclines of pore size and water volume that yield equivalent amounts of Carp eDNA. 4.Our results suggest that aqueous macrobial eDNA predominantly exists inside mitochondria or cells, and that settling plays an important role in its fate. For optimal eDNA capture, we recommend 0.2 !m filtration or a combination of larger pore size and water volume that exceeds the 0.2 !m isocline. In situ filtration of large volumes could maximize detection probability . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx

Summary1. Using environmental DNA (eDNA) to detect aquatic macroorganisms is a new survey method with broad applicability. However, the origin, state and fate of aqueous macrobial eDNA -which collectively determine how well eDNA can serve as a proxy for directly observing organisms and how eDNA should be captured, purified and assayed -are poorly understood. 2. The size of aquatic particles provides clues about their origin, state and fate. We used sequential filtration size fractionation to measure the particle size distribution (PSD) of macrobial eDNA, specifically Common Carp (hereafter referred to as Carp) eDNA. We compared it to the PSDs of total eDNA (from all organisms) and suspended particle matter (SPM). We quantified Carp mitochondrial eDNA using a custom qPCR assay, total eDNA with fluorometry and SPM with gravimetric analysis. 3. In a lake and a pond, we found Carp eDNA in particles from >180 to <0Á2 lm, but it was most abundant from 1 to 10 lm. Total eDNA was most abundant below 0Á2 lm, and SPM was most abundant above 100 lm. SPM consisted of ≤0Á1% total eDNA, and total eDNA consisted of ≤0Á0004% Carp eDNA. 0Á2 lm filtration maximized Carp eDNA capture (85% AE 6%) while minimizing total (i.e. non-target) eDNA capture (48% AE 3%), but filter clogging limited this pore size to a sample volume <250 mL. To mitigate this limitation, we estimated a continuous PSD model for Carp eDNA and derived an equation for calculating isoclines of pore size and water volume that yield equivalent amounts of Carp eDNA. 4. Our results suggest that aqueous macrobial eDNA predominantly exists inside mitochondria or cells, and that settling may therefore play an important role in its fate. For optimal eDNA capture, we recommend 0Á2 lm filtration or a combination of larger pore size and water volume that exceeds the 0Á2 lm isocline. In situ filtration of large volumes could maximize detection probability when surveying large habitats for rare organisms. Our method for eDNA particle size analysis enables future research to compare the PSDs of eDNA from other organisms and environments, and to easily apply them for ecological monitoring.

Adaptive evolution in new or changing environments can be difficult to predict because the functional connections between genotype, phenotype, and fitness are complex. Here, we make these explicit connections by combining field and laboratory experiments in wild mice. We first directly estimate natural selection on pigmentation traits and an underlying pigment locus, Agouti, by using experimental enclosures of mice on different soil colors. Next, we show how a mutation in Agouti associated with survival causes lighter coat color through changes in its protein binding properties. Together, our findings demonstrate how a sequence variant alters phenotype and then reveal the ensuing ecological consequences that drive changes in population allele frequency, thereby illuminating the process of evolution by natural selection.

Metabarcoding of vertebrate DNA derived from carrion flies has been proposed as a promising tool for biodiversity monitoring. To evaluate its efficacy, we conducted metabarcoding surveys of carrion flies on Barro Colorado Island (BCI), Panama, which has a well-known mammal community, and compared our results against diurnal transect counts and camera trapping. We collected 1,084 flies in 29 sampling days, conducted metabarcoding with mammal-specific (16S) and vertebrate-specific (12S) primers, and sequenced amplicons on Illumina MiSeq. For taxonomic assignment, we compared blast with the new program protax, and we found that protax improved species identifications. We detected 20 mammal, four bird, and one lizard species from carrion fly metabarcoding, all but one of which are known from BCI. Fly metabarcoding detected more mammal species than concurrent transect counts (29 sampling days, 13 species) and concurrent camera trapping (84 sampling days, 17 species), and detected 67% of the number of mammal species documented by 8 years of transect counts and camera trapping combined, although fly metabarcoding missed several abundant species. This study demonstrates that carrion fly metabarcoding is a powerful tool for mammal biodiversity surveys and has the potential to detect a broader range of species than more commonly used methods.