Refining analyses of existing data sets is valuable for macrogenetics: a response to Paz-Vinas, Jensen et al., (2021).

Paz-Vinas, Jensen et al. (2021) comment on data and methodological limits of Millette, Fugère, Debyser et al. (2020)-some affect a small proportion of our data sets and analyses and others need to be tackled more generally. These points do not refute our main conclusion of no strong signal of human impacts on COI variation globally.

Resistance, resilience, and functional redundancy of freshwater bacterioplankton communities facing a gradient of agricultural stressors in a mesocosm experiment.

Agricultural pollution with fertilizers and pesticides is a common disturbance to freshwater biodiversity. Bacterioplankton communities are at the base of aquatic food webs, but their responses to these potentially interacting stressors are rarely explored. To test the extent of resistance and resilience in bacterioplankton communities faced with agricultural stressors, we exposed freshwater mesocosms to single and combined gradients of two commonly used pesticides: the herbicide glyphosate (0-15 mg/L) and the neonicotinoid insecticide imidacloprid (0-60 µg/L), in high or low nutrient backgrounds. Over the 43-day experiment, we tracked variation in bacterial density with flow cytometry, carbon substrate use with Biolog EcoPlates, and taxonomic diversity and composition with environmental 16S rRNA gene amplicon sequencing. We show that only glyphosate (at the highest dose, 15 mg/L), but not imidacloprid, nutrients, or their interactions measurably changed community structure, favouring members of the Proteobacteria including the genus Agrobacterium. However, no change in carbon substrate use was detected throughout, suggesting functional redundancy despite taxonomic changes. We further show that communities are resilient at broad, but not fine taxonomic levels: 24 days after glyphosate application the precise amplicon sequence variants do not return, and tend to be replaced by phylogenetically close taxa. We conclude that high doses of glyphosate - but still within commonly acceptable regulatory guidelines - alter freshwater bacterioplankton by favouring a subset of higher taxonomic units (i.e., genus to phylum) that transiently thrive in the presence of glyphosate. Longer-term impacts of glyphosate at finer taxonomic resolution merit further investigation.

Widespread agrochemicals differentially affect zooplankton biomass and community structure.

Anthropogenic environmental change is causing habitat deterioration at unprecedented rates in freshwater ecosystems. Despite increasing more rapidly than many other agents of global change, synthetic chemical pollution-including agrochemicals such as pesticides-has received relatively little attention in freshwater community and ecosystem ecology. Determining the combined effects of multiple agrochemicals on complex biological systems remains a major challenge, requiring a cross-field integration of ecology and ecotoxicology. Using a large-scale array of experimental ponds, we investigated the response of zooplankton community properties (biomass, composition, and diversity metrics) to the individual and joint presence of three globally widespread agrochemicals: the herbicide glyphosate, the neonicotinoid insecticide imidacloprid, and nutrient fertilizers. We tracked temporal variation in zooplankton biomass and community structure along single and combined pesticide gradients (each spanning eight levels), under low (mesotrophic) and high (eutrophic) nutrient-enriched conditions, and quantified (1) response threshold concentrations, (2) agrochemical interactions, and (3) community resistance and recovery. We found that the biomass of major zooplankton groups differed in their sensitivity to pesticides: ≥0.3 mg/L glyphosate elicited long-lasting declines in rotifer communities, both pesticides impaired copepods (≥3 µg/L imidacloprid and ≥5.5 mg/L glyphosate), whereas some cladocerans were highly tolerant to pesticide contamination. Strong interactive effects of pesticides were only recorded in ponds treated with the combination of the highest doses. Overall, glyphosate was the most influential driver of aggregate community properties of zooplankton, with biomass and community structure responding rapidly but recovering unequally over time. Total community biomass showed little resistance when first exposed to glyphosate, but rapidly recovered and even increased with glyphosate concentration over time; in contrast, taxon richness decreased in more contaminated ponds but failed to recover. Our results indicate that the biomass of tolerant taxa compensated for the loss of sensitive species after the first exposure, conferring greater community resistance upon a subsequent contamination event; a case of pollution-induced community tolerance in freshwater animals. These findings suggest that zooplankton biomass may be more resilient to agrochemical pollution than community structure; yet all community properties measured in this study were affected at glyphosate concentrations below common water quality guidelines in North America.

Human influences on the strength of phenotypic selection.

Human activities are driving rapid phenotypic change in many species, with harvesting considered to be a particularly potent evolutionary force. We hypothesized that faster evolutionary change in human-disturbed populations could be caused by a strengthening of phenotypic selection, for example, if human disturbances trigger maladaptation and/or increase the opportunity for selection. We tested this hypothesis by synthesizing 1,366 phenotypic selection coefficients from 37 species exposed to various anthropogenic disturbances, including harvest. We used a paired design that only included studies measuring selection on the same traits in both human-disturbed and control (not obviously human-disturbed "natural") populations. Surprisingly, this meta-analysis did not reveal stronger selection in human-disturbed environments; in fact, we even found some evidence that human disturbances might slightly reduce selection strength. The only clear exceptions were two fisheries showing very strong harvest selection. On closer inspection, we discovered that many disturbances weakened selection by increasing absolute fitness and by decreasing the opportunity for selection-thus explaining what initially seemed a counterintuitive result. We discuss how human disturbances can sometimes weaken rather than strengthen selection, and why measuring the total effect of disturbances on selection is exceedingly difficult. Despite these challenges, documenting human influences on selection can reveal disturbances with particularly strong effects (e.g., fishing), and thus better inform the management of populations exposed to these disturbances.

No consistent effects of humans on animal genetic diversity worldwide.

Human impacts on genetic diversity are poorly understood yet critical to biodiversity conservation. We used 175 247 COI sequences collected between 1980 and 2016 to assess the global effects of land use and human density on the intraspecific genetic diversity of 17 082 species of birds, fishes, insects and mammals. Human impacts on mtDNA diversity were taxon and scale-dependent, and were generally weak or non-significant. Spatial analyses identified weak latitudinal diversity gradients as well as negative effects of human density on insect diversity, and negative effects of intensive land use on fish diversity. The observed effects were predominantly associated with species turnover. Time series analyses found nearly an equal number of positive and negative temporal trends in diversity, resulting in no net monotonic trend in diversity over this time period. Our analyses reveal critical data and theory gaps and call for increased efforts to monitor global genetic diversity.

Trophic structure modulates community rescue following acidification.

Community rescue occurs when a community that experiences lethal stress persists only through the spread of rare types, either genotypes or species, resistant to the stress. Rescue interacts with trophic structure because physical stress experienced by a focal assemblage within the community may also be experienced by its predators and prey. In general, trophic structure will facilitate rescue only when a stress has a less severe effect on a focal assemblage than on its predators. In other circumstances, when stress affects prey or has only a weak effect on predators, trophic structure is likely to hamper rescue. We exposed a community of phytoplankton and zooplankton derived from a natural lake to acidification in outdoor mesocosms large enough to support trophically complex communities. Rescue of the phytoplankton from severe acidification was facilitated by prior exposure to sublethal stress, confirming previous results from microcosm experiments. Even communities that have previously been less highly stressed were eventually rescued, however, because their zooplankton predators were more sensitive to acidification and became extinct. Our experiment shows how community rescue following severe stress is modulated by the differential effect of the stress relative to trophic level.

Sickness behaviour associated with non-lethal infections in wild primates.

Non-lethal parasite infections are common in wildlife, but there is little information on their clinical consequences. Here, we pair infection data from a ubiquitous soil-transmitted helminth, the whipworm (genus Trichuris), with activity data from a habituated group of wild red colobus monkeys (Procolobus rufomitratus tephrosceles) in Kibale National Park, Uganda. We use mixed-effect models to examine the relationship between non-lethal parasitism and red colobus behaviour. Our results indicate that red colobus increased resting and decreased more energetically costly behaviours when shedding whipworm eggs in faeces. Temporal patterns of behaviour also changed, with individuals switching behaviour less frequently when whipworm-positive. Feeding frequency did not differ, but red colobus consumption of bark and two plant species from the genus Albizia, which are used locally in traditional medicines, significantly increased when animals were shedding whipworm eggs. These results suggest self-medicative plant use, although additional work is needed to verify this conclusion. Our results indicate sickness behaviours, which are considered an adaptive response by hosts during infection. Induction of sickness behaviour in turn suggests that these primates are clinically sensitive to non-lethal parasite infections.

Relationship between stream size, watershed land use, and pesticide concentrations in headwater streams.

A quantitative multiresidue study of current-use pesticides in multiple matrices was undertaken with field sampling at 32 headwater streams near Lac Saint-Pierre in Québec, Canada. A total of 232 samples were collected in five campaigns of stream waters and streambed sediments from streams varying in size and watershed land use. Novel multiresidue analytical methods from previous work were successfully applied for the extraction of pesticide residues from sediments via pressurized liquid extraction (PLE) and quantitative analysis using ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) with online sample preparation on a hydrophilic-lipophilic balance (HLB) column. Of the 31 target compounds, including 29 pesticides and two degradation products of atrazine, 29 compounds were detected at least once. Consistent with other studies, atrazine and metolachlor were the most widely-detected herbicides. Detections were generally higher in water than sediment samples and the influence of land use on pesticide concentrations was only detectable in water samples. Small streams with a high proportion of agricultural land use in their watershed were generally found to have the highest pesticide concentrations. Corn and soybean monoculture crops, specifically, were found to cause the greatest impact on pesticide concentration in headwater streams and correlated strongly with many of the most frequently detected pesticides. This study highlights the importance of performing multiresidue pesticide monitoring programs in headwater streams in order to capture the impacts of agricultural intensification on freshwater ecosystems.

A Glyphosate-Based Herbicide Cross-Selects for Antibiotic Resistance Genes in Bacterioplankton Communities.

Agrochemicals often contaminate freshwater bodies, affecting microbial communities that underlie aquatic food webs. For example, the herbicide glyphosate has the potential to indirectly select for antibiotic-resistant bacteria. Such cross-selection could occur if the same genes (encoding efflux pumps, for example) confer resistance to both glyphosate and antibiotics. To test for cross-resistance in natural aquatic bacterial communities, we added a glyphosate-based herbicide (GBH) to 1,000-liter mesocosms filled with water from a pristine lake. Over 57 days, we tracked changes in bacterial communities with shotgun metagenomic sequencing and annotated metagenome-assembled genomes (MAGs) for the presence of known antibiotic resistance genes (ARGs), plasmids, and resistance mutations in the enzyme targeted by glyphosate (enolpyruvyl-shikimate-3-phosphate synthase; EPSPS). We found that high doses of GBH significantly increased ARG frequency and selected for multidrug efflux pumps in particular. The relative abundance of MAGs after a high dose of GBH was predictable based on the number of ARGs in their genomes (17% of variation explained) and, to a lesser extent, by resistance mutations in EPSPS. Together, these results indicate that GBHs can cross-select for antibiotic resistance in natural freshwater bacteria. IMPORTANCE Glyphosate-based herbicides (GBHs) such as Roundup formulations may have the unintended consequence of selecting for antibiotic resistance genes (ARGs), as demonstrated in previous experiments. However, the effects of GBHs on ARGs remain unknown in natural aquatic communities, which are often contaminated with pesticides from agricultural runoff. Moreover, the resistance provided by ARGs compared to canonical mutations in the glyphosate target enzyme, EPSPS, remains unclear. Here, we performed a freshwater mesocosm experiment showing that a GBH strongly selects for ARGs, particularly multidrug efflux pumps. These selective effects were evident after just a few days, and the ability of bacteria to survive and thrive after GBH stress was predictable by the number of ARGs in their genomes and, to a lesser extent, by mutations in EPSPS. Intensive GBH application may therefore have the unintended consequence of selecting for ARGs in natural freshwater communities.

Electrical signalling of dominance in a wild population of electric fish.

Animals often use signals to communicate their dominance status and avoid the costs of combat. We investigated whether the frequency of the electric organ discharge (EOD) of the weakly electric fish, Sternarchorhynchus sp., signals the dominance status of individuals. We correlated EOD frequency with body size and found a strong positive relationship. We then performed a competition experiment in which we found that higher frequency individuals were dominant over lower frequency ones. Finally, we conducted an electrical playback experiment and found that subjects more readily approached and attacked the stimulus electrodes when they played low-frequency signals than high-frequency ones. We propose that EOD frequency communicates dominance status in this gymnotiform species.

Regulation of radial glial motility by visual experience.

Radial glia in the developing optic tectum express the key guidance molecules responsible for topographic targeting of retinal axons. However, the extent to which the radial glia are themselves influenced by retinal inputs and visual experience remains unknown. Using multiphoton live imaging of radial glia in the optic tectum of intact Xenopus laevis tadpoles in conjunction with manipulations of neural activity and sensory stimuli, radial glia were observed to exhibit spontaneous calcium transients that were modulated by visual stimulation. Structurally, radial glia extended and retracted many filopodial processes within the tectal neuropil over minutes. These processes interacted with retinotectal synapses and their motility was modulated by nitric oxide (NO) signaling downstream of neuronal NMDA receptor (NMDAR) activation and visual stimulation. These findings provide the first in vivo demonstration that radial glia actively respond both structurally and functionally to neural activity, via NMDAR-dependent NO release during the period of retinal axon ingrowth.

Community rescue in experimental phytoplankton communities facing severe herbicide pollution.

Community rescue occurs when ecological or evolutionary processes restore positive growth in a highly stressful environment that was lethal to the community in its ancestral form, thus averting biomass collapse in a deteriorating environment. Laboratory evidence suggests that community rescue is most likely in high-biomass communities that have previously experienced moderate doses of sublethal stress. We assessed this result under more natural conditions, in a mesocosm experiment with phytoplankton communities exposed to the ubiquitous herbicide glyphosate. We tested whether community biomass and prior herbicide exposure would facilitate community rescue after severe contamination. We found that prior exposure to glyphosate was a very strong predictor of the rescue outcome, while high community biomass was not. Furthermore, although glyphosate had negative effects on diversity, it did not influence community composition significantly, suggesting a modest role for genus sorting in this rescue process. Our results expand the scope of community rescue theory to complex ecosystems and confirm that prior stress exposure is a key predictor of rescue.

Species differences in group size and electrosensory interference in weakly electric fishes: implications for electrosensory processing.

In animals with active sensory systems, group size can have dramatic effects on the sensory information available to individuals. In "wave-type" weakly electric fishes there is a categorical difference in sensory processing between solitary fish and fish in groups: when conspecifics are within about 1m of each other, the electric fields mix and produce interference patterns that are detected by electroreceptors on each individual. Neural circuits in these animals must therefore process two streams of information-salient signals from prey items and predators and social signals from nearby conspecifics. We investigated the parameters of social signals in two genera of sympatric weakly electric fishes, Apteronotus and Sternopygus, in natural habitats of the Napo River valley in Ecuador and in laboratory settings. Apteronotus were most commonly found in pairs along the Napo River (47% of observations; maximum group size 4) and produced electrosensory interference at rates of 20-300 Hz. In contrast, Sternopygus were alone in 80% of observations (maximum group size 2) in the same region of Ecuador. Similar patterns were observed in laboratory experiments: Apteronotus were in groups and preferentially approached conspecific-like signals in an electrotaxis experiment whereas Sternopygus tended to be solitary and did not approach conspecific-like electrosensory signals. These results demonstrate categorical differences in social electrosensory-related activation of central nervous system circuits that may be related to the evolution of the jamming avoidance response that is used in Apteronotus but not Sternopygus to increase the frequency of electrosensory interference patterns.