Urban birds become less fearful following COVID-19 reopenings.

Following the COVID-19 pandemic, many people around the world stayed home, drastically altering human activity in cities. This exceptional moment provided researchers the opportunity to test how urban animals respond to human disturbance, in some cases testing fundamental questions on the mechanistic impact of urban behaviours on animal behaviour. However, at the end of this 'anthropause', human activity returned to cities. How might each of these strong shifts affect wildlife in the short and long term? We focused on fear response, a trait essential to tolerating urban life. We measured flight initiation distance-at both individual and population levels-for an urban bird before, during and after the anthropause to examine if birds experienced longer-term changes after a year and a half of lowered human presence. Dark-eyed juncos did not change fear levels during the anthropause, but they became drastically less fearful afterwards. These surprising and counterintuitive findings, made possible by following the behaviour of individuals over time, has led to a novel understanding that fear response can be driven by plasticity, yet not habituation-like processes. The pandemic-caused changes in human activity have shown that there is great complexity in how humans modify a behavioural trait fundamental to urban tolerance in animals.

Rapid evolutionary divergence of a songbird population following recent colonization of an urban area.

Colonization of a novel environment by a few individuals can lead to rapid evolutionary change, yet there is scarce evidence of the relative contributions of neutral and selective factors in promoting divergence during the early stages of colonization. Here we explore the role of neutral and selective forces in the divergence of a unique urban population of the dark-eyed junco (Junco hyemalis), which became established on the campus of the University of California at San Diego (UCSD) in the early 1980s. Previous studies based on microsatellite loci documented significant genetic differentiation of the urban population as well as divergence in phenotypic traits relative to nearby montane populations, yet the geographical origin of the colonization and the contributing factors remained uncertain. Our genome-wide single nucleotide polymorphism data set confirmed the marked genetic differentiation of the UCSD population, and we identified the coastal subspecies pinosus from central California as its sister group instead of the neighbouring mountain population. Demographic inference recovered a separation from pinosus as recent as 20-32 generations ago after a strong bottleneck, suggesting a role for drift in genetic differentiation. However, we also found significant associations between habitat variables and genome-wide variants linked to functional genes, some of which have been reported as potentially adaptive in birds inhabiting modified environments. These results suggest that the interplay between founder events and selection may result in rapid shifts in neutral and adaptive loci across the genome, and reveal the UCSD junco population as a case of contemporary evolutionary divergence in an anthropogenic environment.

Using a newly introduced framework to measure ecological stressor interactions.

Understanding how stressors combine to affect population abundances and trajectories is a fundamental ecological problem with increasingly important implications worldwide. Generalisations about interactions among stressors are challenging due to different categorisation methods and how stressors vary across species and systems. Here, we propose using a newly introduced framework to analyse data from the last 25 years on ecological stressor interactions, for example combined effects of temperature, salinity and nutrients on population survival and growth. We contrast our results with the most commonly used existing method - analysis of variance (ANOVA) - and show that ANOVA assumptions are often violated and have inherent limitations for detecting interactions. Moreover, we argue that rescaling - examining relative rather than absolute responses - is critical for ensuring that any interaction measure is independent of the strength of single-stressor effects. In contrast, non-rescaled measures - like ANOVA - find fewer interactions when single-stressor effects are weak. After re-examining 840 two-stressor combinations, we conclude that antagonism and additivity are the most frequent interaction types, in strong contrast to previous reports that synergy dominates yet supportive of more recent studies that find more antagonism. Consequently, measuring and re-assessing the frequency of stressor interaction types is imperative for a better understanding of how stressors affect populations.

Nests in the cities: adaptive and non-adaptive phenotypic plasticity and convergence in an urban bird.

Phenotypic plasticity plays a critical role in adaptation to novel environments. Behavioural plasticity enables more rapid responses to unfamiliar conditions than evolution by natural selection. Urban ecosystems are one such novel environment in which behavioural plasticity has been documented. However, whether such plasticity is adaptive, and if plasticity is convergent among urban populations, is poorly understood. We studied the nesting biology of an 'urban-adapter' species, the dark-eyed junco (Junco hyemalis), to understand the role of plasticity in adapting to city life. We examined (i) whether novel nesting behaviours are adaptive, (ii) whether pairs modify nest characteristics in response to prior outcomes, and (iii) whether two urban populations exhibit similar nesting behaviour. We monitored 170 junco nests in urban Los Angeles and compared our results with prior research on 579 nests from urban San Diego. We found that nests placed in ecologically novel locations (off-ground and on artificial surfaces) increased fitness, and that pairs practiced informed re-nesting in site selection. The Los Angeles population more frequently nested off-ground than the San Diego population and exhibited a higher success rate. Our findings suggest that plasticity facilitates adaptation to urban environments, and that the drivers behind novel nesting behaviours are complex and multifaceted.

When more is less: Emergent suppressive interactions in three-drug combinations.

BACKGROUND: In drug-drug interactions, there are surprising cases in which the growth inhibition of bacteria by a single antibiotic decreases when a second antibiotic is added. These interactions are termed suppressive and have been argued to have the potential to limit the evolution of resistance. Nevertheless, little attention has been given to suppressive interactions because clinical studies typically search for increases in killing efficiency and because suppressive interactions are believed to be rare based on pairwise studies. RESULTS: Here, we quantify the effects of single-, double-, and triple-drug combinations from a set of 14 antibiotics and 3 bacteria strains, totaling 364 unique three-drug combinations per bacteria strain. We find that increasing the number of drugs can increase the prevalence of suppressive interactions: 17% of three-drug combinations are suppressive compared to 5% of two-drug combinations in this study. Most cases of suppression we find (97%) are "hidden" cases for which the triple-drug bacterial growth is less than the single-drug treatments but exceeds that of a pairwise combination. CONCLUSIONS: We find a surprising number of suppressive interactions in higher-order drug combinations. Without examining lower-order (pairwise) bacterial growth, emergent suppressive effects would be missed, potentially affecting our understanding of evolution of resistance and treatment strategies for resistant pathogens. These findings suggest that careful examination of the full factorial of drug combinations is needed to uncover suppressive interactions in higher-order combinations.

Complex patterns of morphological diversity across multiple populations of an urban bird species.

Urbanization presents a natural evolutionary experiment because selection pressures in cities can be strongly mismatched with those found in species' historic habitats. However, some species have managed to adapt and even thrive in these novel conditions. When a species persists across multiple cities, a fundamental question arises: do we see similar traits evolve in similar novel environments? By testing if and how similar phenotypes emerge across multiple urban populations, we can begin to assess the predictability of population response to anthropogenic change. Here, we examine variation within and across multiple populations of a songbird, the dark-eyed junco (Junco hyemalis). We measured morphological variations in juncos across urban and nonurban populations in Southern California. We investigated whether the variations we observed were due to differences in environmental conditions across cities. Bill shape differed across urban populations; Los Angeles and Santa Barbara juncos had shorter, deeper bills than nonurban juncos, but San Diego juncos did not. On the other hand, wing length decreased with the built environment, regardless of the population. Southern Californian urban juncos exhibit both similarities and differences in morphological traits. Studying multiple urban populations can help us determine the predictability of phenotypic evolutionary responses to novel environments.

Evolution Under Thermal Stress Affects Escherichia coli's Resistance to Antibiotics.

Exposure to both antibiotics and temperature changes can induce similar physiological responses in bacteria. Thus, changes in growth temperature may affect antibiotic resistance. Previous studies have found that evolution under antibiotic stress causes shifts in the optimal growth temperature of bacteria. However, little is known about how evolution under thermal stress affects antibiotic resistance. We examined 100+ heat-evolved strains of Escherichia coli that evolved under thermal stress. We asked whether evolution under thermal stress affects optimal growth temperature, if there are any correlations between evolving in high temperatures and antibiotic resistance, and if these strains' antibiotic efficacy changes depending on the local environment's temperature. We found that: (1) surprisingly, most of the heat-evolved strains displayed a decrease in optimal growth temperature and overall growth relative to the ancestor strain, (2) there were complex patterns of changes in antibiotic resistance when comparing the heat-evolved strains to the ancestor strain, and (3) there were few significant correlations among changes in antibiotic resistance, optimal growth temperature, and overall growth.

Meta-analysis of three-stressor combinations on population-level fitness reveal substantial higher-order interactions.

Although natural populations are typically subjected to multiple stressors, most past research has focused on single-stressor and two-stressor interactions, with little attention paid to higher-order interactions among three or more stressors. However, higher-order interactions increasingly appear to be widespread. Consequently, we used a recently introduced and improved framework to re-analyze higher-order ecological interactions. We conducted a literature review of the last 100 years (1920-2020) and reanalyzed 142 ecological three-stressor interactions on species' populations from 38 published papers; the vast majority of these studies were from the past 10 years. We found that 95.8 % (n = 136) of the three-stressor combinations had either not been categorized before or resulted in different interactions than previously reported. We also found substantial levels of emergent properties-interactions that are not due to strong pairwise interactions within the combination but rather uniquely due to all three stressors being combined. Calculating net interactions-the overall accounting for all possible interactions within a combination including the emergent and all pairwise interactions-we found that the most prevalent interaction type is antagonism, corresponding to a smaller than expected effect based on single stressor effects. In contrast, for emergent interactions, the most prevalent interaction type is synergistic, resulting in a larger than expected effect based on single stressor effects. Additionally, we found that hidden suppressive interactions-where a pairwise interaction is suppressed by a third stressor-are found in the majority of combinations (74 %). Collectively, understanding multiple stressor interactions through applying an appropriate framework is crucial for answering fundamental questions in ecology and has implications for conservation biology and population management. Crucially, identifying emergent properties can reveal hidden suppressive interactions that could be particularly important for the ecological management of at-risk populations.

The evolution of resistance to synergistic multi-drug combinations is more complex than evolving resistance to each individual drug component.

Multidrug antibiotic resistance is an urgent public health concern. Multiple strategies have been suggested to alleviate this problem, including the use of antibiotic combinations and cyclic therapies. We examine how adaptation to (1) combinations of drugs affects resistance to individual drugs, and to (2) individual drugs alters responses to drug combinations. To evaluate this, we evolved multiple strains of drug resistant Staphylococcus epidermidis in the lab. We show that evolving resistance to four highly synergistic combinations does not result in cross-resistance to all of their components. Likewise, prior resistance to one antibiotic in a combination does not guarantee survival when exposed to the combination. We also identify four 3-step and four 2-step treatments that inhibit bacterial growth and confer collateral sensitivity with each step, impeding the development of multidrug resistance. This study highlights the importance of considering higher-order drug combinations in sequential therapies and how antibiotic interactions can influence the evolutionary trajectory of bacterial populations.

No evidence of repeated song divergence across multiple urban and non-urban populations of dark-eyed juncos (Junco hyemalis) in Southern California.

Urbanization can affect species communication by introducing new selection pressures, such as noise pollution and different environmental transmission properties. These selection pressures can trigger divergence between urban and non-urban populations. Songbirds rely on vocalizations to defend territories and attract mates. Urban songbirds have been shown in some species and some populations to increase the frequencies, reduce the length and change other temporal features of their songs. This study compares songs from four urban and three non-urban populations of dark-eyed juncos (Junco hyemalis) throughout Southern California. We examined song length, trill rate, minimum frequency, maximum frequency, peak frequency and frequency bandwidth. We also compared songs recorded from one urban junco population in San Diego nearly two decades ago with recently collected data in 2018-2020. Over all comparisons, we only found significant differences between UCLA and the 2006/2007 UCSD field seasons in minimum and maximum frequencies. These findings partially support and are partially in contrast to previous urban song studies. As urban areas expand, more opportunities will arise to understand urban song divergence in greater detail.

Drug interactions modulate the potential for evolution of resistance.

Antimicrobial treatments increasingly rely on multidrug combinations, in part because of the emergence and spread of antibiotic resistance. The continued effectiveness of combination treatments depends crucially on the frequency with which multidrug resistance arises. Yet, it is unknown how this propensity for resistance depends on cross-resistance and on epistatic interactions-ranging from synergy to antagonism-between the drugs. Here, we analyzed how interactions between pairs of drugs affect the spontaneous emergence of resistance in the medically important pathogen Staphylococcus aureus. Resistance is selected for within a window of drug concentrations high enough to inhibit wild-type growth but low enough for some resistant mutants to grow. Introducing an experimental method for high-throughput colony imaging, we counted resistant colonies arising across a two-dimensional matrix of drug concentrations for each of three drug pairs. Our data show that these different drug combinations have significantly different impacts on the size of the window of drug concentrations where resistance is selected for. We framed these results in a mathematical model in which the frequencies of resistance to single drugs, cross-resistance, and epistasis combine to determine the propensity for multidrug resistance. The theory suggests that drug pairs which interact synergistically, preferred for their immediate efficacy, may in fact favor the future evolution of resistance. This framework reveals the central role of drug epistasis in the evolution of resistance and points to new strategies for combating the emergence of drug-resistant bacteria.

Antibiotics Shift the Temperature Response Curve of Escherichia coli Growth.

Temperature variation-through time and across climatic gradients-affects individuals, populations, and communities. Yet how the thermal response of biological systems is altered by environmental stressors is poorly understood. Here, we quantify two key features-optimal temperature and temperature breadth-to investigate how temperature responses vary in the presence of antibiotics. We use high-throughput screening to measure growth of Escherichia coli under single and pairwise combinations of 12 antibiotics across seven temperatures that range from 22°C to 46°C. We find that antibiotic stress often results in considerable changes in the optimal temperature for growth and a narrower temperature breadth. The direction of the optimal temperature shifts can be explained by the similarities between antibiotic-induced and temperature-induced damage to the physiology of the bacterium. We also find that the effects of pairs of stressors in the temperature response can often be explained by just one antibiotic out of the pair. Our study has implications for a general understanding of how ecological systems adapt and evolve to environmental changes. IMPORTANCE The growth of living organisms varies with temperature. This dependence is described by a temperature response curve that is described by an optimal temperature where growth is maximized and a temperature range (termed breadth) across which the organism can grow. Because an organism's temperature response evolves or acclimates to its environment, it is often assumed to change over only evolutionary or developmental timescales. Counter to this, we show here that antibiotics can quickly (over hours) change the optimal growth temperature and temperature breadth for the bacterium Escherichia coli. Moreover, our results suggest a shared-damage hypothesis: when an antibiotic damages similar cellular components as hot (or cold) temperatures do, this shared damage will combine and compound to more greatly reduce growth when that antibiotic is administered at hot (or cold) temperatures. This hypothesis could potentially also explain how temperature responses are modified by stressors other than antibiotics.

Evolutionary consequences of feedbacks between within-host competition and disease control.

Lay Summary: Competition often occurs among diverse parasites within a single host, but control efforts could change its strength. We examined how the interplay between competition and control could shape the evolution of parasite traits like drug resistance and disease severity.

Stressor interaction networks suggest antibiotic resistance co-opted from stress responses to temperature.

Environmental factors like temperature, pressure, and pH partly shaped the evolution of life. As life progressed, new stressors (e.g., poisons and antibiotics) arose as part of an arms race among organisms. Here we ask if cells co-opted existing mechanisms to respond to new stressors, or whether new responses evolved de novo. We use a network-clustering approach based purely on phenotypic growth measurements and interactions among the effects of stressors on population growth. We apply this method to two types of stressors-temperature and antibiotics-to discover the extent to which their cellular responses overlap in Escherichia coli. Our clustering reveals that responses to low and high temperatures are clearly separated, and each is grouped with responses to antibiotics that have similar effects to cold or heat, respectively. As further support, we use a library of transcriptional fluorescent reporters to confirm heat-shock and cold-shock genes are induced by antibiotics. We also show strains evolved at high temperatures are more sensitive to antibiotics that mimic the effects of cold. Taken together, our results strongly suggest that temperature stress responses have been co-opted to deal with antibiotic stress.

Variation in Mutant Prevention Concentrations.

Objectives:Understanding how phenotypic traits vary has been a longstanding goal of evolutionary biologists. When examining antibiotic-resistance in bacteria, it is generally understood that the minimum inhibitory concentration (MIC) has minimal variation specific to each bacterial strain-antibiotic combination. However, there is a less studied resistance trait, the mutant prevention concentration (MPC), which measures the MIC of the most resistant sub-population. Whether and how MPC varies has been poorly understood. Here, we ask a simple, yet important question: How much does the MPC vary, within a single strain-antibiotic association? Using a Staphylococcus species and five antibiotics from five different antibiotic classes-ciprofloxacin, doxycycline, gentamicin, nitrofurantoin, and oxacillin-we examined the frequency of resistance for a wide range of concentrations per antibiotic, and measured the repeatability of the MPC, the lowest amount of antibiotic that would ensure no surviving cells in a 1010 population of bacteria. Results: We found a wide variation within the MPC and distributions that were rarely normal. When antibiotic resistance evolved, the distribution of the MPC changed, with all distributions becoming wider and some multi-modal. Conclusion: Unlike the MIC, there is high variability in the MPC for a given bacterial strain-antibiotic combination.

Phenotypic plasticity and the evolution of a socially selected trait following colonization of a novel environment.

Novel selection pressures in new environments arise through two distinct processes. First, environmental conditions directly affect the fitness of different phenotypes. Second, phenotypic plasticity alters the distribution of phenotypes, thereby placing populations in new selective regimes. A small isolated population of dark-eyed juncos Junco hyemalis became established in San Diego, probably in the early 1980s and probably from the nearby mountains. The relatively mild coastal climate has resulted in an increase in both the mean and the variance of the length of time females breed each year, and this is assumed to be a result of phenotypic plasticity. The population has evolved reduced white in the tail. We studied contemporary patterns of selection on tail white, in the context of the altered breeding season length. Late-hatched nestlings had higher survival and were in better condition than early-hatched nestlings, but among survivors, late-hatched birds had less tail white. We suggest this reflects juvenile mortality favoring individuals with less tail white. In adults, we found weak sexual selection and no viability selection but positive selection on female tail white in association with fecundity. We argue that altered breeding season length had a major impact on patterns of selection and evolution in this population.

Intermediate Levels of Antibiotics May Increase Diversity of Colony Size Phenotype in Bacteria.

Antibiotics select for resistant bacteria whose existence and emergence is more likely in populations with high phenotypic and genetic diversity. Identifying the mechanisms that generate this diversity can thus have clinical consequences for drug-resistant pathogens. We show here that intermediate levels of antibiotics are associated with higher levels of phenotypic diversity in size of colony forming units (cfus), within a single bacterial population. We examine experimentally thousands of populations of bacteria subjected to different disturbance levels that are created by varying antibiotic concentrations. Based on colony sizes, we find that intermediate levels of antibiotics always result in the highest phenotypic variation of this trait. This result is supported across bacterial densities and in the presence of three different antibiotics with two different mechanisms of action. Our results suggest intermediate levels of a stressor (as opposed to very low or very high levels) could affect the phenotypic diversity of a population, at least with regards to the single trait measured here. While this study is limited to a single phenotypic trait within a single species, the results suggest examining phenotypic and genetic variation created by disturbances and stressors could be a promising way to understand and limit variation in pathogens.

Patterns of Bird-Bacteria Associations.

Birds, with their broad geographic ranges and close association with humans, have historically played an important role as carriers of human disease and as reservoirs for drug-resistant bacteria. Here, we examine scientific literature over a 15-year timespan to identify reported avian-bacterial associations and factors that may impact zoonotic disease emergence by classifying traits of bird species and their bacteria. We find that the majority of wild birds studied were migratory, in temperate habitats, and in the order Passeriformes. The highest diversity of bacteria was found on birds in natural habitats. The most frequently reported bacteria were Escherichia coli, Salmonella enterica, and Campylobacter jejuni. Of the bacteria species reported, 54% have shown pathogenicity toward humans. Percentage-wise, more pathogens were found in tropical (vs. temperate) habitats and natural (vs. suburban, urban, or agricultural) habitats. Yet, only 22% were tested for antibiotic resistance, and of those tested, 75% of bacteria species were resistant to at least one antibiotic. There were no significant patterns of antibiotic resistance in migratory versus non-migratory birds, temperate versus tropical areas, or different habitats. We discuss biases in detection and representation, and suggest a need for increased sampling in non-temperate zones and in a wider range of avian species.

Prevalence and patterns of higher-order drug interactions in Escherichia coli.

Interactions and emergent processes are essential for research on complex systems involving many components. Most studies focus solely on pairwise interactions and ignore higher-order interactions among three or more components. To gain deeper insights into higher-order interactions and complex environments, we study antibiotic combinations applied to pathogenic Escherichia coli and obtain unprecedented amounts of detailed data (251 two-drug combinations, 1512 three-drug combinations, 5670 four-drug combinations, and 13608 five-drug combinations). Directly opposite to previous assumptions and reports, we find higher-order interactions increase in frequency with the number of drugs in the bacteria's environment. Specifically, as more drugs are added, we observe an elevated frequency of net synergy (effect greater than expected based on independent individual effects) and also increased instances of emergent antagonism (effect less than expected based on lower-order interaction effects). These findings have implications for the potential efficacy of drug combinations and are crucial for better navigating problems associated with the combinatorial complexity of multi-component systems.

Suppressive drug combinations and their potential to combat antibiotic resistance.

Antibiotic effectiveness often changes when two or more such drugs are administered simultaneously and unearthing antibiotic combinations with enhanced efficacy (synergy) has been a longstanding clinical goal. However, antibiotic resistance, which undermines individual drugs, threatens such combined treatments. Remarkably, it has emerged that antibiotic combinations whose combined effect is lower than that of at least one of the individual drugs can slow or even reverse the evolution of resistance. We synthesize and review studies of such so-called 'suppressive interactions' in the literature. We examine why these interactions have been largely disregarded in the past, the strategies used to identify them, their mechanistic basis, demonstrations of their potential to reverse the evolution of resistance and arguments for and against using them in clinical treatment. We suggest future directions for research on these interactions, aiming to expand the basic body of knowledge on suppression and to determine the applicability of suppressive interactions in the clinic.