Connectivity at the human-wildlife interface: starling movements relate to carriage of E. coli.

Synanthropic bird species in human, poultry or livestock environments can increase the spread of pathogens and antibiotic-resistant bacteria between wild and domestic animals. We present the first telemetry-based spatial networks for a small songbird. We quantified landscape connectivity exerted by spotless starling movements, and aimed to determine if connectivity patterns were related to carriage of potential pathogens. We captured 28 starlings on a partridge farm in 2020 and tested them for Avian influenza virus, West Nile virus WNV, Avian orthoavulavirus 1, Coronavirus, Salmonella spp. and Escherichia coli. We did not detect any viruses or Salmonella, but one individual had antibodies against WNV or cross-reacting Flaviviruses. We found E. coli in 61 % (17 of 28) of starlings, 76 % (13 of 17) of which were resistant to gentamicin, 12 % (2 of 17) to cefotaxime/enrofloxacin and 6 % (1 of 17) were phenotypic extended spectrum beta-lactamase (ESBL) carriers. We GPS-tracked 17 starlings and constructed spatial networks showing how their movements (i.e. links) connect different farms with nearby urban and natural habitats (i.e. nodes with different attributes). Using E. coli carriage as a proxy for acquisition/dispersal of bacteria, we found differences across spatial networks constructed for E. coli positive (n = 7) and E. coli negative (n = 9) starlings. We used Exponential Random Graph Models to reveal significant differences between networks. In particular, an urban roost was more connected to other sites by movements of E. coli positive than by movements of E. coli negative starlings. Furthermore, an open pine forest used mainly for roosting was more connected to other sites by movements of E. coli negative than by movements of E. coli positive starlings. Using E. coli as a proxy for a potential pathogen carried by starlings, we reveal the pathways of spread that starlings could provide between farms, urban and natural habitats.

Male starling floaters preferentially visit nests of males with reduced resource holding potential.

Floaters are sexually mature individuals that are not able to reproduce by defending breeding resources. Floaters often visit active nests, probably to gather public information or to compete for a nesting site. We tested the hypothesis that floaters preferentially prospect nests in which they have a better chance of taking over, and that they do so by assessing the owners' resource holding potential (RHP). We manipulated the flight capacity of male and female breeders in a population of spotless starlings (Sturnus unicolor) by clipping two flight feathers per wing before egg laying, thus increasing their wing-load and likely impairing their condition. We subsequently monitored breeder and floater activity by means of transponder readers during the nestling period. We found that nests owned by wing-clipped males were visited by a greater number of male floaters than control nests. This effect was absent in the case of wing-clipped females. The number of male floaters also increased with increasing nestling age and number of parental visits. The experiment shows that male floaters preferentially prospect nests in which the owner shows a reduced RHP, a strategy that likely allows them to evict weak owners and take over their nests for future reproductive attempts.

Concordance of movements and songs enhances receiver responses to multimodal display in the starling.

Many animals produce signals that consist of vocalizations and movements to attract mates or deter rivals. We usually consider them as components of a single multimodal signal because they are temporally coordinated. Sometimes, however, this relationship takes on a more complex spatiotemporal character, resembling choreographed music. Timing is important for audio-visual integration, but choreographic concordance requires even more skill and competence from the signaller. Concordance should therefore have a strong impact on receivers; however, little is known about its role in audio-visual perception during natural interactions. We studied the effects of movement and song type concordance in audio-visual displays of the starling, Sturnus vulgaris. Starlings produce two types of movements that naturally appear in specific phrases of songs with a similar temporal structure and amplitude. In an experiment with a taxidermic robotic model, males responded more to concordant audio-visual displays, which are also naturally preferred, than to discordant displays. In contrast, the effect of concordance was independent of the specific combination of movement and song types in a display. Our results indicate that the concordance of movements and songs was critical to the efficacy of the display and suggest that the information that birds gained from concordance could not be obtained by adding information from movements and songs.

The genome of a globally invasive passerine, the common myna, Acridotheres tristis.

In an era of global climate change, biodiversity conservation is receiving increased attention. Conservation efforts are greatly aided by genetic tools and approaches, which seek to understand patterns of genetic diversity and how they impact species health and their ability to persist under future climate regimes. Invasive species offer vital model systems in which to investigate questions regarding adaptive potential, with a particular focus on how changes in genetic diversity and effective population size interact with novel selection regimes. The common myna (Acridotheres tristis) is a globally invasive passerine and is an excellent model species for research both into the persistence of low-diversity populations and the mechanisms of biological invasion. To underpin research on the invasion genetics of this species, we present the genome assembly of the common myna. We describe the genomic landscape of this species, including genome wide allelic diversity, methylation, repeats, and recombination rate, as well as an examination of gene family evolution. Finally, we use demographic analysis to identify that some native regions underwent a dramatic population increase between the two most recent periods of glaciation, and reveal artefactual impacts of genetic bottlenecks on demographic analysis.

Recent beak evolution in North American starlings after invasion.

European starlings are one of the most abundant and problematic avian invaders in the world. From their native range across Eurasia and North Africa, they have been introduced to every continent except Antarctica. In 160 years, starlings have expanded into different environments throughout the world, making them a powerful model for understanding rapid evolutionary change and adaptive plasticity. Here, we investigate their spatiotemporal morphological variation in North America and the native range. Our dataset includes 1217 specimens; a combination of historical museum skins and modern birds. Beak length in the native range has remained unchanged during the past 206 years, but we find beak length in North American birds is now 8% longer than birds from the native range. We discuss potential drivers of this pattern including dietary adaptation or climatic pressures. Additionally, body size in North American starlings is smaller than those from the native range, which suggests a role for selection or founder effect. Taken together, our results indicate rapid recent evolutionary change in starling morphology coincident with invasion into novel environments.

Elevated lead (Pb) in urban European starling (Sturnus vulgaris) feathers is not correlated to physiology or behavior.

Urbanization is rapidly changing the environment and creating new challenges in the lives of animals across the globe. Anthropogenic contaminants-like heavy metals-can persist within the environment for prolonged periods of time and present a widespread problem for those living near contaminated areas. Lead (Pb) was a commonly used heavy metal that continues to threaten the health of all organisms despite being phased out, especially in urban areas where historical use was more common. In this study, a common urban-adapter, the European starling (Sturnus vulgaris), was trapped to explore whether feather Pb burden is greater in birds from urban habitats than rural habitats, as well as whether Pb burdens were correlated with behavior, physiology, and feather development. Across four sites (two rural and two urban), soil Pb concentrations were measured and 197 free-living starlings were captured to measure feather Pb concentrations. Using linear mixed models, this study found that urban starling nestlings had elevated feather Pb burdens compared to rural nestlings. In contrast, there was no correlation between Pb and urbanization in adult birds whose exposure to Pb may reflect a larger spatial range compared to nestlings. For both nestlings and adults, feather Pb was uncorrelated to corticosterone, testosterone, aggressive behavior, or feather growth rates. These findings suggest that starlings may be a useful biomonitoring tool to detect Pb in the local environment, however, the age and spatial range of birds is a critical consideration in applying this tool. Further work is needed to understand the intricate relationship between heavy metals, behavior, morphological development, and physiology in free-living organisms.

Phenotypic flexibility in metabolic adjustments and digestive function in white-shouldered starlings: responses to short-term temperature acclimation.

Changing the intrinsic rate of metabolic heat production is the main adaptive strategy for small birds to cope with different ambient temperatures. In this study, we tested the hypothesis that the small passerine the white-shouldered starling (Sturnus sinensis) can modulate basal metabolism under temperature acclimation by changing the morphological, physiological and biochemical state of its tissues and organs. We measured the effects of temperature on body mass, basal metabolic rate (BMR), wet mass of various internal organs, state 4 respiration (S4R) and cytochrome c oxidase (CCO) activity in the pectoral muscle and organs, metabolites in the pectoral muscle, energy intake, histological dynamics and the activity of duodenal digestive enzymes. Warm acclimation decreased BMR to a greater extent than cold acclimation. At the organ level, birds in the cold-acclimated group had significantly heavier intestines but significantly lighter pectoral muscles. At the cellular level, birds in the cold-acclimated group showed significantly higher S4R in the liver and heart and CCO activity in the liver and kidney at both the mass-specific and whole-organ levels. A metabolomic analysis of the pectoral tissue revealed significantly higher lipid decomposition, amino acid degradation, ATP hydrolysis, and GTP and biotin synthesis in cold-acclimated birds. Acclimation to cold significantly increased the gross energy intake (GEI), feces energy (FE) and digestive energy intake (DEI) but significantly decreased the digestive efficiency of these birds. Furthermore, cold-acclimated birds had a higher maltase activity and longer villi in the duodenum. Taken together, these data show that white-shouldered starlings exhibit high phenotypic flexibility in metabolic adjustments and digestive function under temperature acclimation, consistent with the notion that small birds cope with the energy challenges presented by a cold environment by modulating tissue function in a way that would affect BMR.

The molecular architecture of a complex social behavior: gregarious song.

The medial preoptic area (mPOA) regulates the probability and intensity of singing behavior in birds. Polzin and colleagues examined the molecular changes in the mPOA that were associated with gregarious song in European starlings (Sturnus vulgaris). High-throughput transcriptome analyses identified glutamate and dopamine pathways were highly enriched with gregarious song.

COMPARISON OF HEMATOLOGIC DIFFERENCES WITH LITHIUM HEPARIN AND DIPOTASSIUM ETHYLENEDIAMINETETRAACETIC ACID IN EUROPEAN STARLINGS (STURNUS VULGARIS).

Preservation of blood through use of anticoagulants allows delayed assessment of hematologic health and is commonly employed in veterinary health assessments. The two most common anticoagulants are lithium heparin (LH) and dipotassium ethylenediaminetetraacetic acid (EDTA), and their effects can vary widely between species. The hematologic effects of these anticoagulants on blood from European starlings (Sturnus vulgaris) have not been established, and these birds could serve as models for passerine species both in managed collections and in the wild. Blood was drawn from 45 European starlings and immediately divided into either LH or EDTA microtainers. For each sample, packed cell volume (PCV), total solids (TS), and complete blood counts were performed. There were no significant differences between EDTA and LH anticoagulated blood for PCV, white blood cell count (WBC) slide estimates, WBC determined by Leukopet, absolute heterophils, absolute lymphocytes, absolute monocytes, absolute eosinophils, or absolute basophils. Blood anticoagulated with EDTA had higher total solids than blood mixed with LH. For both anticoagulants, Leukopet-measured total WBC were consistently higher than blood film estimates. There were no subjective morphologic differences for WBC and no hemolysis observed in the samples. Thrombocyte clumping was prominent for LH blood samples and minimal for EDTA samples. These results reveal that LH and EDTA are both suitable anticoagulants for use in European starlings, and EDTA may be superior for diagnostic purposes or for qualitative evaluation of thrombocyte quantity.

Distinct patterns of activity within columns of the periaqueductal gray are associated with functionally distinct birdsongs.

Male songbirds produce female-directed songs in spring that convey a state of sexual motivation. Many songbirds also sing in fall flocks in affiliative/gregarious contexts in which song is linked to an intrinsic positive affective state. The periaqueductal gray (PAG) in mammals, which is organized into functional columns, integrates information from multiple brain regions and relays this information to vocal motor areas so that an animal emits a vocal signal reflective of its affective state. Here, we test the hypothesis that distinct columns in the songbird PAG play roles in the distinct affective states communicated by sexually motivated and gregarious song. We quantified the numbers of immediate early gene ZENK-positive cells in 16 PAG subregions in male European starlings (Sturnus vulgaris) after singing gregarious or sexually motivated song. Results suggest that distinct PAG columns in songbirds context-specifically regulate song, agonistic, and courtship behaviors. A second exploratory, functional tract-tracing study also demonstrated that inputs to the PAG from specific subregions of the medial preoptic nucleus may contribute to gregarious song and behaviors indicative of social dominance. Together, findings suggest that conserved PAG columns and inputs from the preoptic nucleus may play a role in context-specific vocal and other social behaviors.

Starling: Introducing a mesoscopic scale with Confluence for Graph Clustering.

Given a Graph G = (V, E) and two vertices i, j ∈ V, we introduce Confluence(G, i, j), a vertex mesoscopic closeness measure based on short Random walks, which brings together vertices from a same overconnected region of the Graph G, and separates vertices coming from two distinct overconnected regions. Confluence becomes a useful tool for defining a new Clustering quality function QConf(G, Γ) for a given Clustering Γ and for defining a new heuristic Starling to find a partitional Clustering of a Graph G intended to optimize the Clustering quality function QConf. We compare the accuracies of Starling, to the accuracies of three state of the art Graphs Clustering methods: Spectral-Clustering, Louvain, and Infomap. These comparisons are done, on the one hand with artificial Graphs (a) Random Graphs and (b) a classical Graphs Clustering Benchmark, and on the other hand with (c) Terrain-Graphs gathered from real data. We show that with (a), (b) and (c), Starling is always able to obtain equivalent or better accuracies than the three others methods. We show also that with the Benchmark (b), Starling is able to obtain equivalent accuracies and even sometimes better than an Oracle that would only know the expected overconnected regions from the Benchmark, ignoring the concretely constructed edges.

Comparative performance of microscopy, nested PCR, and real-time PCR for screening avian haemosporidian parasites in Afrotropical starlings (family Sturnidae).

Prevalence studies of avian haemosporidian parasites frequently use microscopy and the nested polymerase chain reaction (PCR) protocols for detecting infections. Newer PCR protocols to detect parasites are being developed, with the distinct advantage of reducing screening cost and time, as well as increasing efficiency and sensitivity. The detection ability of microscopy and nested PCR was compared against a real-time PCR (qPCR) protocol using genomic DNA extracted from 240 bird blood samples collected from three starling species (Cape Starling, the Greater Blue-eared Starling, and the Wattled Starling; family Sturnidae) in the Kruger national park, South Africa. All three protocols successfully detected avian haemosporidian parasites with the qPCR having a considerable edge against the other two methods. Fifteen unique cytochrome b lineages were identified of which seven were new lineages. Microscopy and nested PCR recorded similar prevalence (32.92% and 35.42% respectively). The qPCR protocol used here, although more sensitive (52.92% prevalence), is not able to differentiate between parasite genera but provides the opportunity to screen a large number of samples in large-scale studies within a specific region. This study recommends the development and adoption of new molecular protocols with increased sensitivity and accuracy in prevalence studies. Nevertheless, microscopy remains essential for the morphological description of parasites and for distinguishing between abortive and successful chronic infections. The PCR-based method displays the detection of the parasitic genome but does not reveal whether parasites have or will develop into a successful infection.

Distinct patterns of gene expression in the medial preoptic area are related to gregarious singing behavior in European starlings (Sturnus vulgaris).

BACKGROUND: Song performed in flocks by European starlings (Sturnus vulgaris), referred to here as gregarious song, is a non-sexual, social behavior performed by adult birds. Gregarious song is thought to be an intrinsically reinforced behavior facilitated by a low-stress, positive affective state that increases social cohesion within a flock. The medial preoptic area (mPOA) is a region known to have a role in the production of gregarious song. However, the neurochemical systems that potentially act within this region to regulate song remain largely unexplored. In this study, we used RNA sequencing to characterize patterns of gene expression in the mPOA of male and female starlings singing gregarious song to identify possibly novel neurotransmitter, neuromodulator, and hormonal pathways that may be involved in the production of gregarious song. RESULTS: Differential gene expression analysis and rank rank hypergeometric analysis indicated that dopaminergic, cholinergic, and GABAergic systems were associated with the production of gregarious song, with multiple receptor genes (e.g., DRD2, DRD5, CHRM4, GABRD) upregulated in the mPOA of starlings who sang at high rates. Additionally, co-expression network analyses identified co-expressing gene clusters of glutamate signaling-related genes associated with song. One of these clusters contained five glutamate receptor genes and two glutamate scaffolding genes and was significantly enriched for genetic pathways involved in neurodevelopmental disorders associated with social deficits in humans. Two of these genes, GRIN1 and SHANK2, were positively correlated with performance of gregarious song. CONCLUSIONS: This work provides new insights into the role of the mPOA in non-sexual, gregarious song in starlings and highlights candidate genes that may play a role in gregarious social interactions across vertebrates. The provided data will also allow other researchers to compare across species to identify conserved systems that regulate social behavior.

Variation in oxidative status, but not structural and physiological development, associated with changing ontogenetic environments.

Despite the potential for temporally dependent relationships between trait values and fitness (e.g. as juveniles approach life-stage transitions such as fledging), how developmental stage affects canalization (a measure of robustness to environmental variation) of morphological and physiological traits is rarely considered. To test the sensitivity of morphological and physiological traits to environmental variation in two developmental stages, we manipulated brood size at hatch in European starlings (Sturnus vulgaris) and cross-fostered chicks between enlarged and reduced broods approaching fledging. We measured body size (mass, tarsus, wing length) and physiological state (aerobic capacity, oxidative status) at asymptotic mass on day 15, then cross-fostered chicks between 'high' and 'low' quality environments and assessed the same traits again on day 20, after 5 days of pre-fledging mass recession. Chicks in reduced broods were heavier at asymptotic mass and had lower reactive oxygen metabolites than enlarged broods, whereas structural size, aerobic capacity and antioxidant capacity were unaffected by experimental brood size. The observed canalization of structural and physiological traits during early development was maintained after cross-fostering, during late development. However, in contrast to early development, antioxidant capacity approaching fledging appeared sensitive to environmental conditions, as trajectories varied by cross-fostering treatment. Elevated reactive oxygen metabolites observed after early development in enlarged brood chicks were maintained after cross-fostering, suggesting that canalized development in low-quality environments could produce oxidative costs that carry over between life stages, even when conditions improve. These data reveal trait-specific relationships between environmental conditions and development, and highlight how natal environment effects may vary by developmental stage.

The motivation to flock correlates with vocal-social behaviors and dopamine-related gene expression in male European starlings.

It is proposed that songbird flocks are partly reinforced by positive social interactions, however not all flock mate interactions are positive. The combination of both positive and negative social interactions with flock mates may play a role in the motivation for birds to flock. The nucleus accumbens (NAc), medial preoptic area (POM), and ventral tegmental area (VTA) are implicated in vocal-social behaviors in flocks, including singing. Dopamine (DA) within these regions modifies motivated, reward-directed behaviors. Here, we begin to test the hypothesis that individual social interactions and DA within these regions are involved in the motivation to flock. Vocal-social behaviors were recorded in eighteen male European starlings in mixed-sex flocks in fall, when starlings are highly social and form large flocks. Males were then singly removed from their flock and the motivation to flock was quantified as the amount of time spent attempting to join a flock following separation. We used quantitative real-time polymerase chain reaction to measure expression of DA-related genes in the NAc, POM, and VTA. Birds producing high levels of vocal behaviors were more highly motivated to flock and had higher tyrosine hydroxylase (the rate-limiting enzyme in DA synthesis) expression in the NAc and VTA. Birds that received high levels of agonistic behaviors were less motivated to flock and had higher DA receptor subtype 1 expression in the POM. Overall, our findings suggest that interplay between social experience and DA activity in NAc, POM, and VTA plays a key role in social motivation in flocking songbirds.

Fluctuations in the Rate of Radioactive Decay of the Natural Potassium-40 Isotope Can Potentially Affect the Periodic "Spontaneous" Activity of Animals.

Body temperature rhythms of C57Bl/6 laboratory mice and common greenfinches (Chloris chloris) and feeding periods of common starlings (Sturnus vulgaris) were compared with the intensity of fluctuations in 40К radioactive decay. Body temperature changes in greenfinches and mice were found to positively correlate with the intensity of fluctuations in 40K radioactive decay. Superposed epoch analysis showed that an increase in mouse body temperature, which reflects the start of the active phase in the sleep-wake cycle, and food intake in starlings coincide with an increase in the intensity of 40K radioactive decay. Thus, animal activity in the ultradian range of periods may be related to external quasi-rhythmic physical influences, rather than being determined only by endogenous processes. Given the extremely low dose of natural 40К exposure, a factor responsible for radioactivity fluctuations may act as a biotropic factor.

Dehydroepiandrosterone (DHEA) increases undirected singing behavior and alters dopaminergic regulation of undirected song in non-breeding male European starlings (Sturnus vulgaris).

Introduction: It has been proposed that in species that defend territories across multiple life history stages, brain metabolism of adrenal dehydroepiandrosterone (DHEA) regulates aggressive behavior at times when gonadal androgen synthesis is low (i.e. the non-breeding season). To date, a role for DHEA in the regulation of other forms of social behavior that are expressed outside of the context of breeding remains unknown. Methods: In this experiment, we used the European starling (Sturnus vulgaris) model system to investigate a role for DHEA in the neuroendocrine regulation of singing behavior by males in non-breeding condition. Starling song in a non-breeding context is spontaneous, not directed towards conspecifics, and functions to maintain cohesion of overwintering flocks. Results: Using within-subjects design, we found that DHEA implants significantly increase undirected singing behavior by non-breeding condition male starlings. Given that DHEA is known to modulate multiple neurotransmitter systems including dopamine (DA) and DA regulates undirected song, we subsequently used immunohistochemistry for phosphorylated tyrosine hydroxylase (pTH, the active form of the rate-limiting enzyme in DA synthesis) to investigate the effect of DHEA on dopaminergic regulation of singing behavior in a non-breeding context. Pearson correlation analysis revealed a positive linear association between undirected singing behavior and pTH immunoreactivity in the ventral tegmental area and midbrain central gray of DHEA-implanted, but not control-implanted, males. Discussion: Taken together, these data suggest that undirected singing behavior by non-breeding starlings is modulated by effects of DHEA on dopaminergic neurotransmission. More broadly, these data expand the social behavior functions of DHEA beyond territorial aggression to include undirected, affiliative social communication.

Tracing the introduction of the invasive common myna using population genomics.

The common myna (Acridotheres tristis) is one of the most invasive bird species in the world, yet its colonisation history is only partly understood. We identified the introduction history and population structure, and quantified the genetic diversity of myna populations from the native range in India and introduced populations in New Zealand, Australia, Fiji, Hawaii, and South Africa, based on thousands of single nucleotide polymorphism markers in 814 individuals. We were able to identify the source population of mynas in several invasive locations: mynas from Fiji and Melbourne, Australia, were likely founded by individuals from a subpopulation in Maharashtra, India, while mynas in Hawaii and South Africa were likely independently founded by individuals from other localities in India. Our findings suggest that New Zealand mynas were founded by individuals from Melbourne, which, in turn, were founded by individuals from Maharashtra. We identified two genetic clusters among New Zealand mynas, divided by New Zealand's North Island's axial mountain ranges, confirming previous observations that mountains and thick forests may form barriers to myna dispersal. Our study provides a foundation for other population and invasion genomic studies and provides useful information for the management of this invasive species.

Prevalence of haemosporidia in Asian Glossy Starling with discovery of misbinding of Haemoproteus-specific primer to Plasmodium genera in Sarawak, Malaysian Borneo.

BACKGROUND: Plasmodium, Haemoproteus and Leucocytozoon are three mainly studied blood parasites known to cause malarial and pseudomalarial infections in avian worldwide. Although Sarawak is a biodiversity hotspot, molecular data on blood parasite diversity in birds are absent. The objective of the study is to determine the prevalence of blood parasite in Asian Glossy Starlings (AGS), an urban bird with high population density in Sarawak and to elucidate the phylogenetic relationship with other blood parasite. METHODS: Twenty-nine carcasses of juvenile AGS that were succumbed to death due to window collision were collected around the vicinity of Universiti Malaysia Sarawak. Nested-multiplex and nested PCR targeting the Cytochrome B gene were used to detect Plasmodium and Haemoproteus, and Leucocytozoon respectively. Two primer sets were used for Haemoproteus detection to increase detection sensitivity, with one being a genus-specific primer. RESULTS: Fourteen samples (prevalence rate: 48.28%) were found positive for avian Plasmodium. Phylogenetic analysis divided our sequences into five lineages, pFANTAIL01, pCOLL4, pACCBAD01, pALPSIS01 and pALPSIS02, with two lineages being novel. No Haemoproteus and Leucocytozoon was found in this study. However, Haemoproteus-specific primer used amplified our Plasmodium samples, making the primer non-specific to Haemoproteus only. CONCLUSION: This is the first blood parasite detection study on AGS using carcasses and blood clot as sample source in Sarawak. Due to the scarcity of longer sequences from regions with high genetic plasticity, usage of genus-specific primers should be validated with sequencing to ensure correct prevalence interpretation.

Midsession reversal task with starlings: A quantitative test of the timing hypothesis.

In the Mid-Session Reversal task (MSR), an animal chooses between two options, S1 and S2. Rewards follow S1 but not S2 from trials 1-40, and S2 but not S1 from trials 41-80. With pigeons, the psychometric function relating S1 choice proportion to trial number starts close to 1 and ends close to 0, with indifference (PSE) close to trial 40. Surprisingly, pigeons make anticipatory errors, choosing S2 before trial 41, and perseverative errors, choosing S1 after trial 40. These errors suggest that they use time into the session as the preference reversal cue. We tested this timing hypothesis with 10 Spotless starlings. After learning the MSR task with a T-s Inter-Trial Interval (ITI), they were exposed to either 2 T or T/2 ITIs during testing. Doubling the ITI should shift the psychometric function to the left and halve its PSE, whereas halving the ITI should shift the function to the right and double its PSE. When the starlings received one pellet per reward, the ITI manipulation was effective: The psychometric functions shifted in the direction and by the amount predicted by the timing hypothesis. However, non-temporal cues also influenced choice.