The use of environmental DNA (eDNA) in biodiversity assessments offers a step-change in sensitivity, throughput and simultaneous measures of ecosystem diversity and function. There remains, however, a need to examine eDNA persistence in the wild through simultaneous temporal measures of eDNA and biota. Here, we use metabarcoding of two markers of different lengths, derived from an annual time series of aqueous lake eDNA to examine temporal shifts in ecosystem biodiversity and in an ecologically important group of macroinvertebrates (Diptera: Chironomidae). The analyses allow different levels of detection and validation of taxon richness and community composition (β-diversity) through time, with shorter eDNA fragments dominating the eDNA community. Comparisons between eDNA, community DNA, taxonomy and UK species abundance data further show significant relationships between diversity estimates derived across the disparate methodologies. Our results reveal the temporal dynamics of eDNA and validate the utility of eDNA metabarcoding for tracking seasonal diversity at the ecosystem scale.

New applications of DNA and RNA sequencing are expanding the field of biodiversity discovery and ecological monitoring, yet questions remain regarding precision and efficiency. Due to primer bias, the ability of metabarcoding to accurately depict biomass of different taxa from bulk communities remains unclear, while PCR-free whole mitochondrial genome (mitogenome) sequencing may provide a more reliable alternative. Here, we used a set of documented mock communities comprising 13 species of freshwater macroinvertebrates of estimated individual biomass, to compare the detection efficiency of COI metabarcoding (three different amplicons) and shotgun mitogenome sequencing. Additionally, we used individual COI barcoding and de novo mitochondrial genome sequencing, to provide reference sequences for OTU assignment and metagenome mapping (mitogenome skimming), respectively. We found that, even though both methods occasionally failed to recover very low abundance species, metabarcoding was less consistent, by failing to recover some species with higher abundances, probably due to primer bias. Shotgun sequencing results provided highly significant correlations between read number and biomass in all but one species. Conversely, the read-biomass relationships obtained from metabarcoding varied across amplicons. Specifically, we found significant relationships for eight of 13 (amplicons B1FR-450 bp, FF130R-130 bp) or four of 13 (amplicon FFFR, 658 bp) species. Combining the results of all three COI amplicons (multiamplicon approach) improved the read-biomass correlations for some of the species. Overall, mitogenomic sequencing yielded more informative predictions of biomass content from bulk macroinvertebrate communities than metabarcoding. However, for large-scale ecological studies, metabarcoding currently remains the most commonly used approach for diversity assessment.

BackgroundInvasive predators may change the structure of invaded communities through predation and competition with native species. In Europe, the invasive signal crayfish Pacifastacus leniusculus is excluding the native white clawed crayfish Austropotamobius pallipes.Methodology and Principal FindingsThis study compared the predatory functional responses and prey choice of native and invasive crayfish and measured impacts of parasitism on the predatory strength of the native species. Invasive crayfish showed a higher (>10%) prey (Gammarus pulex) intake rate than (size matched) natives, reflecting a shorter (16%) prey handling time. The native crayfish also showed greater selection for crustacean prey over molluscs and bloodworm, whereas the invasive species was a more generalist predator. A. pallipes parasitised by the microsporidian parasite Thelohania contejeani showed a 30% reduction in prey intake. We suggest that this results from parasite-induced muscle damage, and this is supported by a reduced (38%) attack rate and increased (30%) prey handling time.Conclusions and SignificanceOur results indicate that the per capita (i.e., functional response) difference between the species may contribute to success of the invader and extinction of the native species, as well as decreased biodiversity and biomass in invaded rivers. In addition, the reduced predatory strength of parasitized natives may impair their competitive abilities, facilitating exclusion by the invader.

1Many crayfish species have been introduced to novel habitats worldwide, often threatening 2 extinction of native species. Here we investigate competitive interactions and parasite infections 3 in the native Austropotamobius pallipes and the invasive Pacifastacus leniusculus from single 4 and mixed species populations in the UK. We found A. pallipes individuals to be significantly 5 smaller in mixed compared to single species populations; conversely P. leniusculus individuals 6were larger in mixed than in single species populations. Our data provide no support for 7 reproductive interference as a mechanism of competitive displacement and instead suggest 8 competitive exclusion of A. pallipes from refuges by P. leniusculus leading to differential 9 predation. We screened fifty-two P. leniusculus and twelve A. pallipes for microsporidian 10 infection using PCR. We present the first molecular confirmation of Thelohania contejeani in the 11 native A. pallipes; in addition, we provide the first evidence for T. contejeani in the invasive P. 12 leniusculus. Three novel parasite sequences were also isolated from P. leniusculus with an 13 overall prevalence of microsporidian infection of 38 % within this species; we discuss the identity 14 of and the similarity between these three novel sequences. We also screened a subset of fifteen 15 P. leniusculus and three A. pallipes for Aphanomyces astaci, the causative agent of crayfish 16 plague and for the protistan crayfish parasite Psorospermium haeckeli. We found no evidence for 17 infection by either agent in any of the crayfish screened. The high prevalence of microsporidian 18 parasites and occurrence of shared T. contejeani infection lead us to propose that future studies 19 should consider the impact of these parasites on native and invasive host fitness and their 20 potential effects upon the dynamics of native-invader systems. 21 22

Non-native species can profoundly affect native ecosystems through trophic interactions with native species. Native prey may respond differently to non-native versus native predators since they lack prior experience. Here we investigate antipredator responses of two common freshwater macroinvertebrates, Gammarus pulex and Potamopyrgus jenkinsi, to olfactory cues from three predators; sympatric native fish (Gasterosteus aculeatus), sympatric native crayfish (Austropotamobius pallipes), and novel invasive crayfish (Pacifastacus leniusculus). G. pulex responded differently to fish and crayfish; showing enhanced locomotion in response to fish, but a preference for the dark over the light in response to the crayfish. P. jenkinsi showed increased vertical migration in response to all three predator cues relative to controls. These different responses to fish and crayfish are hypothesised to reflect the predators' differing predation types; benthic for crayfish and pelagic for fish. However, we found no difference in response to native versus invasive crayfish, indicating that prey naiveté is unlikely to drive the impacts of invasive crayfish. The Predator Recognition Continuum Hypothesis proposes that benefits of generalisable predator recognition outweigh costs when predators are diverse. Generalised responses of prey as observed here will be adaptive in the presence of an invader, and may reduce novel predators' potential impacts. RÉSUMÉ Réponses de macro-invertébrés aquatiques aux prédateurs indigènes et non indigènes Mots-clés :