Abstract Behaviour innovation plays a pivotal role in a species’ adaptability to dynamic environments. Investigating innovative behaviour and its underlying mechanism is therefore crucial for elucidating the development of cognitive flexibility across animals. Animal personality—which shapes how individuals perceive and engage with their surroundings—could offer insights into individual variation in this process. This study used a three-step foraging puzzle to evaluate the innovation capacity in 28 rosy-faced lovebirds ( Agapornis roseicollis ), specifically examining their capacity to recombine individually innovated component behaviours into integrated, more sophisticated techniques. We found that nearly half of the individuals spontaneously innovated multiple component behaviours to solve novel puzzles. Crucially, when challenged with a more sophisticated task, they recombined these behaviours into functionally dependent sequences without prior social demonstration. We further identified sex, persistence, and asocial learning capacity as key predictors of innovative problem-solving performance, with females, persistent individuals, and superior asocial learners excelled at problem-solving. Our findings demonstrate that behavioural innovation is not a static event, but a dynamic process—modulated by physical, cognitive, and personality variables—in which behaviours are flexibly transferred and recombined into increasingly complex forms to enable rapid individual adaptation.

Mosquito control is essential for public health, but the ecological impacts of larvicides on non-target organisms, particularly amphibians, remain poorly understood. In Hong Kong, three larvicides are commonly used: Bacillus thuringiensis israelensis (Bti), temephos, and larvicidal oil. This study evaluated the lethal and sublethal effects of these larvicides on the endangered Romer’s Tree Frog ( Liuixalus romeri ), which occurs in peri-urban habitats in Hong Kong. We assessed the effects of prolonged exposure (up to 86 days) at recommended application rates on survival, growth, and movement behavior in tadpoles, and determined LC₅₀ (lethal concentration that kills 50% of population) values. Under prolonged exposure, larvicidal oil caused near-complete mortality within 21 days and significantly reduced growth and movement. In contrast, Bti and temephos did not affect survival or growth, although both induced sublethal changes in movement behavior. These patterns were consistent with acute toxicity results: larvicidal oil exhibited the highest mortality, with a 96-hour LC₅₀ only 4.6-fold higher than the recommended rate, whereas Bti and temephos showed larger safety margins, with 96-hour LC₅₀ values 53–105-fold and 17–42-fold higher than recommended rates, respectively. Overall, larvicidal oil poses a high ecological risk and should be avoided in amphibian habitats, whereas Bti and temephos represent comparatively lower-risk alternatives. Furthermore, our results demonstrate that exposure duration strongly influences toxicity outcomes. Ecological risk assessments should incorporate both acute and prolonged exposure, as well as lethal and sublethal endpoints, when evaluating impacts of pesticides on non-target organisms.

Sexual dichromatism, characterized by sex-specific differences in coloration, is widespread among birds and often involves carotenoid-based pigmentation. Despite extensive research on the social and ecological environments favoring sexual dichromatism, the molecular mechanisms underlying its development and evolution remain largely unexplored. In this study, we investigated the genetic and molecular processes giving rise to sexual dichromatism in the red ketocarotenoid-based plumage of northern cardinals (Cardinalis cardinalis). We quantified carotenoid concentrations in plasma and feather follicles, confirmed that homologs of CYP2J19, BDH1L, and TTC39B catalyze the production of C-4 ketocarotenoids, and performed gene expression analyses across tissues. Males showed significantly higher plasma and feather ketocarotenoid concentrations, upregulated CYP2J19 and TTC39B expression in liver and feather tissues, and upregulated carotenoid transport gene expression in the gut and feather follicles. Females exhibited a dramatic upregulation of BCO2 in their feather follicles, facilitating carotenoid degradation and attenuating red pigmentation. Additionally, sex-biased expression of hormonal regulators, such as HSD17B4 and ZNF131, in the feather follicle suggests hormonal modulation influences dichromatism. These findings indicate that sex-specific regulation of carotenoid processing genes underpins the vivid red coloration in males and the drab phenotype in females, likely maintained by a balance between natural and sexual selection, with mechanisms of sexual antagonism affecting divergent gene expression. This work advances understanding of the molecular basis of avian sexual dimorphism, highlighting key genetic pathways involved in carotenoid-based coloration and providing a foundation for further research into the evolution of sexually dichromatic traits.

Five new species of Methocha Latreille, 1804 are described from Hong Kong: two species based on females only, Methocha haaksek Barthélémy & Terayama sp. nov. and Methocha leleji Barthélémy & Terayama sp. nov.; two on males only, M. wilsoni Barthélémy & Terayama sp. nov. and M. zetetes Barthélémy & Terayama sp. nov.; and one with females and males associated through molecular analysis, M. hongkongensis Barthélémy & Terayama sp. nov. A key to species and a phylogenetic tree of Methocha occurring in the Hong Kong SAR are provided.

High-sugar diets cause human metabolic diseases, yet several bird lineages convergently adapted to feeding on sugar-rich nectar or fruits. We investigated the underlying molecular mechanisms in hummingbirds, parrots, honeyeaters, and sunbirds by generating nine new genomes and 90 tissue-specific transcriptomes. Comparative screens revealed an excess of repeated selection in both protein-coding and regulatory sequences in sugar-feeding birds, suggesting reuse of genetic elements. Sequence or expression changes in sugar-feeders affect genes involved in blood pressure regulation and lipid, amino acid, and carbohydrate metabolism, with experiments showing functional changes in honeyeater hexokinase 3. MLXIPL, a key regulator of sugar and lipid homeostasis, showed convergent sequence and regulatory changes across all sugar-feeding clades; experiments revealed enhanced sugar-induced transcriptional activity of hummingbird MLXIPL, highlighting its adaptive role in high-sugar diets.

Population declines may have long-term genetic consequences, including genetic erosion and inbreeding depression, which could affect species' evolutionary potential and increase their risk of extinction. Small populations are more vulnerable to genetic threats than common species, but even species with large populations can also be at risk of extinction. The yellow-breasted bunting (Emberiza aureola) is a common and widespread songbird in the northern Palearctic regions, but its global population size has drastically declined by around 90% throughout the past 30 years, leading to an upgrade of its conservation status to critically endangered in the IUCN Red List. In this study, we identified three populations within this species using whole-genome resequencing data, but the genetic differentiation between populations was shallow. These populations underwent similar population fluctuations but differed in the extent of population decline, resulting in lower genetic diversity and more homozygous deleterious mutations in a population comprising individuals on islands. The ancient demographic history was mainly associated with the climate, while population declines over the past 100 generations are likely due to human activities. Our results suggest that the yellow-breasted bunting population before the recent collapse faced relatively low genetic threats and had high evolutionary potential. However, we should be vigilant about the genetic threats faced by this species, as our sampling time occurred at the onset of its recent global population collapse. This study provides valuable genetic information for the conservation of yellow-breasted bunting and also highlights the similar genetic threats faced by other large populations.

The East Asian-Australasian Flyway (EAAF) is experiencing notable population declines in its migratory waterbird species. Understanding the foraging ecology of these waterbirds, including ducks, is crucial for monitoring and safeguarding their food sources and wetland habitats. Here, we used a DNA metabarcoding approach to analyze fecal DNA from duck species to elucidate their dietary composition during the wintering period in a subtropical East Asian wetland. By employing multiple markers (18S, COI, and trnL) targeting different taxonomic groups and levels, we offered a comprehensive dietary analysis for omnivores that consume both plants and animals. We revealed the dietary compositions of common migratory duck species and their intraspecific and interspecific dietary variations. While ducks are generally known to be omnivorous, Anas crecca (green-winged teal) had a more specialized diet and was primarily herbivorous throughout winter. In contrast, the sympatric Mareca penelope (Eurasian wigeon) and Spatula clypeata (northern shoveler) exhibited more omnivorous foraging behaviors. Moreover, A. crecca displayed less dietary variation among samples, while samples of M. penelope and S. clypeata were highly variable in their compositions. Comparing our results with those of studies conducted in different regions, we found that the dietary compositions of these duck species varied to different degrees across geographic locations. This variation underscores the flexibility of these duck species in their diets and their adaptable foraging strategies. Our findings also indicate that grasslands rich in herbaceous plants and aquatic environments abundant with small aquatic invertebrates are vital foraging habitats for duck species during their winter period.

Gene duplication and loss play an important role in the evolution of the major histocompatibility complex (MHC). Variations in copy number and sequence diversity of MHC genes can have significant fitness consequences. Here, we characterized both MHC class I and class II genes in a group of parrots—lovebirds (Agapornis spp.) using cloning and sequencing, quantitative PCR, and depth-of-coverage (DoC) analysis with whole-genome re-sequencing data. We identified copy number variation in MHC class II genes, with A. roseicollis having a single MHCIIB gene copy, whereas A. canus possesses at least three gene copies. Conversely, the copy number of class I genes is invariable, with only one copy identified in each Agapornis species. Phylogenetic reconstructions revealed both concerted evolution and trans-species polymorphism of MHC genes. In both MHC class I and II genes, sequences from the recently diverged eye-ringed species (e.g., A. fischeri, A. personatus, and A. nigrigenis) and their sister species A. roseicollis showed an intercalating pattern with no species-specific clustering, consistent with trans-species polymorphism. In contrast, sequences from the early-diverged species (e.g., A. canus and A. pullarius) clustered by species, which is typical for avian MHC genes undergoing concerted evolution. The pattern of MHC copy number variation and modes of evolution observed are associated with the timescale of species divergence. We suggest that future studies should include both MHC class I and II genes and multiple species spanning a range of divergence time to enhance our understanding of the evolution of avian MHC diversity.

Most bats are insectivorous, but some species have evolved the ability to prey on fish. Although piscivory has been confirmed in the Rickett's big-footed myotis (Myotis pilosus), the extent of piscivory of other cohabiting Myotis species is uncertain. This study aims to explore the dietary niches and fish consumption of three Myotis species in a subtropical East Asian region, and specifically the fish diet of M. pilosus. Our findings reveal, for the first time, that M. pilosus consumes marine fishes, in contrast to previous research conducted in inland regions that suggested year-round consumption of cyprinids in freshwater habitats. We also observed seasonal variation in the diets of M. pilosus. It predominately hunted wide-banded hardyhead silverside [31% relative read abundance (RRA) of all 12S reads], sailfin flying fish, and shorthead anchovy during the wet season, while mainly preying upon mullets (31%) during the dry months. In more inland areas, M. pilosus was found to primarily feed on invasive freshwater poeciliids (13%). Furthermore, M. pilosus consumed more fish during the dry season, while there was a greater consumption of insects during the wet months. Most notably among our findings is the consumption of fish by two individuals of Horsfield's myotis (M. horsfieldii), indicating that the species is potentially piscivorous. We revealed that both M. horsfieldii and M. pilosus consumed water striders, suggesting that foraging of aquatic insects could be driving the evolution of fishing behavior. Our findings have also shed light on the flexibility of foraging behavior in piscivorous bats.

Olfaction and vision can play important roles in optimizing foraging decisions of birds, enabling them to maximize their net rate of energy intake while searching for, handling and consuming food. Parrots have been used extensively in avian cognition research, and some species use olfactory cues to find food. Here we used machine-learning analysis and pose estimation with convolutional neural networks (CNNs) to elucidate the relative importance of visual and olfactory cues for informing foraging decisions in the rosy-faced lovebird, Agapornis roseicollis, as a nontypical model species. In a binary choice experiment, we used markerless body pose tracking to analyse bird response behaviours. Rosy-faced lovebirds quickly learnt to discriminate the feeder provisioned with food by forming an association with visual (red/green papers) but not olfactory (banana/almond odour) cues. When visual cues indicated the provisioned and empty feeders, feeder choice was more successful, hesitation time shorter and interest in the empty feeder significantly lower. Our findings reveal that lovebirds can rapidly learn novel visual cues but not olfactory cues, indicating that vision plays a more important role in their learning and foraging decisions than olfaction. (c) 2025 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Cognitive capacity for associative learning and quantity discrimination is highly adaptive in various ecological contexts and subject to convergent evolution across diverse animal species. Discrimination accuracy generally increases with the ratio between two quantities in many studied animals; however, this ability is expected to vary among species, highlighting the need to understand how it operates in different taxa. Parrots are among the most intelligent birds, but only a few parrot species have been studied for their associative learning and quantity discrimination abilities. To investigate these cognitive capabilities in small parrots, we presented a novel symbol system to 28 rosy-faced lovebirds, Agapornis roseicollis. This system associates additive tally marks with symbols representing a one-to-one correspondence with different food quantities. We specifically tested three aspects of cognition related to numerical competence, namely associative learning, inference and quantity discrimination. Trained lovebirds could spontaneously infer the relative food quantities represented by other symbols. Lovebirds proved capable of (1) associating symbols (i.e. object-file symbolism) with (2) 'more-less' quantity inference by deducing food quantities based on their knowledge of this symbol-quantity association and (3) enhancing their performance in relation to disparity ratio (conforming to Weber's law) and absolute difference. Furthermore, (4) the influence of food ratios and absolute differences varied with different ratio ranges. Within a small ratio range (<= 3), increasing the ratio or absolute difference enhanced discrimination performance. However, within a higher ratio range (>3), these characteristics had less of an impact. We concluded that rosy-faced lovebirds are capable of advanced associative learning and quantity discrimination, similar to larger parrot species. (c) 2025 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Although high-sugar diets are associated with metabolic diseases in humans, several bird lineages have independently evolved to primarily subsist on simple sugars from flower nectar or fruits. In this study, we address a key question of the repeatability of molecular evolution by investigating the convergent and lineage-specific molecular mechanisms underlying dietary adaptations in four major sugar-consuming bird lineages: hummingbirds, parrots, honeyeaters, and sunbirds. We assembled nine new genomes for sugar-consuming species and their closely related non-sugar feeding outgroup species and generated 90 tissue-specific transcriptomes for six key species. We identified signatures of positive selection in both protein-coding and non-coding regulatory sequences, and found positive selection targets the same genes more frequently in sugar-feeders compared to non-sugar feeding controls, suggesting that adapting to a high-sugar diet requires changes in a limited number of genetic elements. At the functional level, pathways associated with energy homeostasis, carbohydrate metabolism, heart function, and hormonal regulation showed convergent selection signals in both protein-coding and regulatory evolution, while lipid and amino acid metabolism demonstrated mostly regulatory evolution. Notably, we observed striking evidence for convergent adaptation in MLXIPL, a transcription factor regulating sugar and lipid homeostasis, manifesting in both sequence and regulatory changes across all sugar-feeders. With functional assays, we demonstrated that hummingbird MLXIPL enhances sugar-induced transcriptional activity in HEK239 cells, suggesting its central role in the evolution of adaptations to high-sugar diets. Our findings elucidate the main genomic targets in the evolution of sugar-feeding at both molecular and pathway levels. ### Competing Interest Statement The authors have declared no competing interest.

The range distributions of many bird species cover extensive geographic distances, exposing each local population to unique ecological challenges. Understanding the molecular basis of how species adapt to diverse habitats across their geographic range is crucial for identifying populations at risk and implementing effective conservation strategies. In this study, we employed two passerine species, the black-capped chickadee (Poecile atricapillus) and the American goldfinch (Spinus tristis), which are widely distributed across North America. This study focused on examining changes in gene expression within their distinct populations inhabiting diverse habitats across various geographical locations. A comparative transcriptomic study was conducted on wild-caught birds from two geographically separate locations, Boston, Massachusetts, and Kent, Ohio, characterized by considerable annual variability in winter severity. We tested the hypothesis that populations of both species in Kent and Boston would show differential gene expression patterns in their brains in response to unique local environmental conditions. Analyzing the differentially expressed genes (DEGs) in black-capped chickadees revealed associations with neural processes such as the generation and maintenance of neurons, activity-dependent plasticity, and cognitive ability. Many of these genes were linked to brain variation in chickadee populations related to spatial cognition associated with food caching. We also compared changes in gene expression levels with coding sequence variability to explore the underlying basis of differential gene expression patterns. We tested the hypothesis that expression differences are driven by underlying genetic variation. A population genetic analysis on transcriptome data from both species revealed no highly divergent genetic variants (single nucleotide polymorphisms or SNPs) in the coding regions of genes identified as differentially expressed. However, some of the DEGs themselves were transcription factors or regulatory molecules, as were some of the genes with highly divergent SNPs. These findings suggest that the genetic architecture underlying the differential gene expression patterns is mostly regulatory rather than protein-coding changes.

Island organisms often evolve phenotypes divergent from their mainland counterparts, providing a useful system for studying adaptation under differential selection. In the white-winged fairywren (Malurus leucopterus), subspecies on two islands have a black nuptial plumage whereas the subspecies on the Australian mainland has a blue nuptial plumage. The black subspecies have a feather nanostructure that could in principle produce a blue structural color, suggesting a blue ancestor. An earlier study proposed independent evolution of melanism on the islands based on the history of subspecies divergence. However, the genetic basis of melanism and the origin of color differentiation in this group are still unknown. Here, we used whole-genome resequencing to investigate the genetic basis of melanism by comparing the blue and black M. leucopterus subspecies to identify highly divergent genomic regions. We identified a well-known pigmentation gene ASIP and four candidate genes that may contribute to feather nanostructure development. Contrary to the prediction of convergent evolution of island melanism, we detected signatures of a selective sweep in genomic regions containing ASIP and SCUBE2 not in the black subspecies but in the blue subspecies, which possesses many derived SNPs in these regions, suggesting that the mainland subspecies has re-evolved a blue plumage from a black ancestor. This proposed re-evolution was likely driven by a preexisting female preference. Our findings provide new insight into the evolution of plumage coloration in island versus continental populations, and, importantly, we identify candidate genes that likely play roles in the development and evolution of feather structural coloration.

Psittacine beak and feather disease virus (BFDV) is a widespread and highly pathogenic virus in parrots. The disease typically presents with feather and beak abnormalities, along with possible immune system suppression. No cure or commercialized vaccine is currently available. Our understanding of the Psittacine beak and feather disease often comes from infected individuals with visible symptoms. Limited knowledge exists regarding the pathology and role of asymptomatic individuals in disease transmission. Asymptomatic individuals could shed the virus in their crop secretion, feces, or feathers. In this study, we investigated the temporal change in the viral load in feather and fecal samples from 17 asymptomatic Rosy-faced Lovebirds (Agapornis roseicollis) using qPCR. Our results showed that most of the individuals had very low viral load, while three individuals with high viral load at the beginning of the experiment were observed to exhibit a decreasing trend in viral load in both fecal and feather samples. Surprisingly, the viral load in an individual can drop from a high level to an undetectable level within three months. This suggests that BFDV infection might not be lethal or highly pathogenic for some individuals. We also showed that the viral load in feathers was higher than in feces.

AbstractPsittacosis, or parrot fever, is a zoonotic disease caused byChlamydiaspecies associated with birds. One of the causative agents of the disease isChlamydia psittaci, which is often carried by psittacine and can be highly pathogenic and virulent to humans. In Hong Kong, a city with high population density, psittacosis is a notifiable disease with over 60% of cases in the last decade resulting in hospitalization. However, the source of transmission ofC. psittaciand its prevalence in pet birds in Hong Kong are currently unknown. To evaluate the risks of psittacosis transmission through pet birds, we tested the presence ofC. psittaciand determined its genotypes in samples obtained from 516 captive birds from households, pet shops, and a veterinary hospital in Hong Kong. Results revealed that five samples (0.97%), collected from budgerigars and cockatiels, wereC. psittaci-positive, while 80% of them were obtained from pet shops. The identified strains belonged to genotype A and were closely related to the strain SP15 that was previously discovered in mainland China. Our study highlights the need for genotypingChlamydiaspecies in psittacosis patients to understand their source(s) of origin.

Parrots have remarkable plumage coloration that result in part from a unique ability to produce pigments called psittacofulvins that yield yellow to red feather colors. Little is known about the evolution of psittacofulvin-based pigmentation. Widespread color mutations of captive-bred parrots provide perfect opportunities to study the genetic basis of this trait. An earlier study on blue budgerigars, which do not possess psittacofulvins, reveals the involvement of an uncharacterized polyketide synthase (MuPKS) in yellow psittacofulvin synthesis. The blue phenotype had repeatedly appeared in different parrot species, similar to independent experimental replications allowing the study of convergent evolution and molecular mechanism of psittacofulvin-based pigmentation. Here, we investigated the genetic basis of the blue phenotypes in two species of Agapornis parrots, Fischer's lovebird (A. fischeri) and Yellow-collared lovebird (A. personatus). Using whole-genome data, we identified a single genomic region with size <2 Mb to be strongly associated with the color difference between blue and wild-type (WT) birds in both species. Surprisingly, we discovered that the mutation associated with the blue Agapornis phenotype was identical to the previously described substitution causing the functional change of MuPKS in budgerigars. Together with the evidence of shared blue-associated haplotypes and signatures of a selective sweep in this genomic region in both species, we demonstrated both de novo mutation and interspecific introgression play a role in the evolution of this trait in different Agapornis species. The convergent substitution in the same gene in both lovebirds and budgerigars also indicates a strong evolutionary constraint on psittacofulvin-based coloration.

Olfaction and vision can play important roles in optimizing foraging decisions of birds, enabling them to maximize their net rate of energy intake while searching for, handling, and consuming food. Parrots have been used extensively in avian cognition research, and some species use olfactory cues to find food. Here we pioneered machine learning analysis and pose-estimation with convolutional neural networks (CNNs) to elucidate the relative importance of visual and olfactory cues for informing foraging decisions in the rosy-faced lovebird ( Agapornis roseicollis ) as a non-typical model species. In a binary choice experiment, we used markerless body pose tracking to analyse bird response behaviours. Rosy-faced lovebirds quickly learnt to discriminate the feeder provisioned with food by forming an association with visual (red/green papers) but not olfactory (banana/almond odour) cues. When visual cues indicated the provisioned and empty feeders, feeder choice was more successful, choice latency shorter, and interest in the empty feeder significantly lower. This demonstrates that visual cues alone are sufficient to inform lovebird foraging decisions without needing to use olfactory cues, suggesting that selection has not driven olfactory-based foraging in lovebird evolution.### Competing Interest StatementThe authors have declared no competing interest.

Chytridiomycosis, an infectious skin disease caused by the chytrid fungi, Batrachochytrium dendrobatidis and B. salamandrivorans, poses a significant threat to amphibian biodiversity worldwide. Antifungal bacteria found on the skin of chytrid-resistant amphibians could potentially provide defense against chytridiomycosis and lower mortality rates among resistant individuals. The Hong Kong newt (Paramesotriton hongkongensis) is native to East Asia, a region suspected to be the origin of chytrids, and has exhibited asymptomatic infection, suggesting a long-term coexistence with the chytrids. Therefore, the skin microbiota of this resistant species warrant investigation, along with other factors that can affect the microbiota. Among the 149 newts sampled in their natural habitats in Hong Kong, China, putative antifungal bacteria were found in all individuals. There were 314 amplicon sequence variants distributed over 25 genera of putative antifungal bacteria; abundant ones included Acinetobacter, Flavobacterium, and Novosphingobium spp. The skin microbiota compositions were strongly influenced by the inter-site geographical distances. Despite inter-site differences, we identified some core skin microbes across sites that could be vital to P. hongkongensis. The dominant cores included the family Comamonadaceae, family Chitinophagaceae, and class Betaproteobacteria. Moreover, habitat elevation and host sex also exhibited significant effects on skin microbiota compositions. The antifungal bacteria found on these newts offer an important resource for conservation against chytridiomycosis, such as developing probiotic treatments for susceptible species.