Widespread gene flow following range expansion in Anna's Hummingbird.

Anthropogenic changes have altered the historical distributions of many North American taxa. As environments shift, ecological and evolutionary processes can combine in complex ways to either stimulate or inhibit range expansion. Here, we examined the role of evolution in a rapid range expansion whose ecological context has been well-documented, Anna's Hummingbird (Calypte anna). Previous studies have suggested that the C. anna range expansion is the result of an ecological release facilitated by human-mediated environmental changes, where access to new food sources have allowed further filling of the abiotic niche. We examined the role of gene flow and adaptation during range expansion from their native California breeding range, north into Canada and east into New Mexico and Texas, USA. Using low coverage whole genome sequencing we found high genetic diversity, low divergence, and little evidence of selection on the northern and eastern expansion fronts. Additionally, there are no clear barriers to gene flow across the native and expanded range. The lack of selective signals between core and expanded ranges could reflect (i) an absence of novel selection pressure in the expanded range (supporting the ecological release hypothesis), (ii) swamping of adaptive variation due to high gene flow, or (iii) limitations of genome scans for detecting small shifts in allele frequencies across many loci. Nevertheless, our results provide an example where strong selection is not apparent during a rapid, contemporary range shift.

Quantifying phenology and migratory behaviours of hummingbirds using single-site dynamics and mark-detection analyses.

Nuanced understanding of seasonal movements of partially migratory birds is paramount to species and habitat conservation. Using nascent statistical methods, we identified migratory strategies of birds outfitted with radio-frequency identification (RFID) tags detected at RFID feeders in two sites in California, USA. We quantified proportions of migrants and residents and the seasonal phenology for each movement strategy in Allen's and Anna's hummingbirds; we also validated our methodology by fitting our model to obligate migratory black-chinned hummingbirds. Allen's and Anna's hummingbirds exhibited characteristics of facultative migratory behaviour. We also quantified apparent annual survival for each migratory strategy and found that residents had significantly higher probabilities of apparent survival. Low survival estimates for migrants suggest that a high proportion of birds in the migrant group permanently emigrated from our study sites. Considered together, our analyses suggest that hummingbirds in both northern and southern California sites partake in diverse and highly plastic migratory behaviours. Our assessment elucidates the dynamics underlying idiosyncratic migratory behaviours of two species of hummingbirds, in addition to describing a framework for similar assessments of migratory behaviours using the multi-state open robust design with state uncertainty model and single-site dynamics.

Evaluation of minimally invasive sampling methods for detecting Avipoxvirus: Hummingbirds as a case example.

Avian pox is a common avian virus that in its cutaneous form can cause characteristic lesions on a bird's dermal surfaces. Detection of avian pox in free-ranging birds historically relied on observations of visual lesions and/or histopathology, both which can underestimate avian pox prevalence. We compared traditional visual observation methods for avian pox with molecular methods that utilize minimally invasive samples (blood, toenail clipping, feathers, and dermal swabs) in an ecologically important group of birds, hummingbirds. Specifically, avian pox prevalence in several species of hummingbirds were examined across multiple locations using three different methods: (1) visual inspection of hummingbirds for pox-like lesions from a long-term banding data set, (2) qPCR assay of samples from hummingbird carcasses from wildlife rehabilitation centers, and (3) qPCR assay of samples from live-caught hummingbirds. A stark difference in prevalences among these three methods was identified, with an avian pox prevalence of 1.5% from banding data, 20.4% from hummingbird carcasses, and 32.5% from live-caught hummingbirds in California. This difference in detection rates underlines the necessity of a molecular method to survey for avian pox, and this study establishes one such method that could be applied to other wild bird species. Across all three methods, Anna's hummingbirds harbored significantly higher avian pox prevalence than other species examined, as did males compared with females and birds caught in Southern California compared with Northern California. After hatch-year hummingbirds also harbored higher avian pox prevalences than hatch-year hummingbirds in the California banding data set and the carcass data set. This is the first study to estimate the prevalence of avian pox in hummingbirds and address the ecology of this hummingbird-specific strain of avian pox virus, providing vital information to inform future studies on this charismatic and ecologically important group of birds.

Retrospective study on admission trends of Californian hummingbirds found in urban habitats (1991-2016).

BACKGROUND: Hummingbirds are frequently presented to California wildlife rehabilitation centers for medical care, accounting for approximately 5% of overall admissions. Age, sex, and reason for admission could impact hummingbird survivability, therefore identification of these factors could help maximize rehabilitation efforts. METHODS: Mixed-effects logistic regression models were used to identify specific threats to the survival of 6908 hummingbirds (1645 nestlings and 5263 non-nestlings) consisting of five species (Calypte anna, Calypte costa, Selasphorus rufus, Selasphorus sasin, Archilochus alexandri), found in urban settings, and admitted to California wildlife rehabilitation centers over 26 years. RESULTS: In total, 36% of birds survived and were transferred to flight cage facilities for further rehabilitation and/or release. Nestlings were more likely to be transferred and/or released compared to adult hummingbirds. After accounting for age, birds rescued in spring and summer were twice as likely to be released compared to birds rescued in the fall. A high number of nestlings were presented to the rehabilitation centers during spring, which coincides with the nesting season for hummingbirds in California, with the lowest number of nestlings presented in fall. Reasons for presentation to rehabilitation centers included several anthropogenic factors such as window collisions (9.6%) and interactions with domesticated animals (12.9%). Survival odds were lower if a hummingbird was rescued in a "torpor-like state" and were higher if rescued for "nest-related" reasons. Evaluation of treatment regimens administered at wildlife rehabilitation centers identified supportive care, including providing commercial nutrient-rich nectar plus solution, to significantly increase hummingbird survivability. DISCUSSION: Our results provide evidence of threats to hummingbirds in urban habitats, based on reasons for rescue and presentation to rehabilitation centers. Reasons for hummingbird admissions to three California wildlife rehabilitation centers were anthropogenic in nature (i.e., being associated with domestic animals, window collisions, and found inside a man-made structure) and constituted 25% of total admissions. There was a clear indication that supportive care, such as feeding a commercial nectar solution, and medical treatment significantly increased the odds of survival for rescued hummingbirds.

Concentrations of Retinol and α-Tocopherol in Tissue Samples From Anna's Hummingbirds (Calypte anna).

Retinol (vitamin A) and α-tocopherol (vitamin E) concentrations were measured in tissue samples (liver, heart, pectoral muscle, and brain) from Anna's Hummingbirds (Calypte anna). Hummingbirds were after-hatch year birds that were sourced from various rehabilitation centers throughout California. Tissues samples were analyzed using high-performance liquid chromatography (HPLC). Minimum, maximum, mean, standard deviation (SD), and median ppm concentrations were calculated for each vitamin and tissue sample type. A novel analytical method was developed to analyze small mass tissue samples, with the smallest sample mass being 0.05 g for which analysis can be performed. Mean ± standard deviation (SD) concentrations of retinol in hummingbird livers, hearts, and pectoral muscle samples were 269.0 ± 216.9 ppm, 1.8 ± 2.2 ppm, and 0.3 ± 0.1 ppm, respectively. Mean ± SD α-tocopherol concentrations were 6.9 ± 4.6 ppm, 5.5 ± 4.0 ppm, 3.7 ± 2.2 ppm, and 9.1 ± 3.2 ppm for liver, heart, pectoral muscle, and brain samples, respectively. Vitamin concentrations from varying tissue types were compared to determine which were best associated with liver concentrations, the most commonly analyzed tissue for these vitamins. For both retinol and α-tocopherol, heart samples were most strongly associated with the liver samples. The results of this study provide baseline retinol and α-tocopherol concentrations in different tissue types from Anna's hummingbirds. These baseline values may be utilized in conservation efforts to avoid hypervitaminosis and hypovitaminosis of rehabilitated and/or captive hummingbirds by providing guidelines for nutritional targets which could be assessed on post-mortem examinations. Post-mortem examination of birds and measurement of vitamin concentrations in tissues may allow for dietary changes that aid captive hummingbirds.

TaqMan quantitative real-time PCR for detecting Avipoxvirus DNA in various sample types from hummingbirds.

BACKGROUND: Avian pox is a viral disease documented in a wide range of bird species. Disease-related detrimental effects can cause dyspnea and dysphagia, and birds with high metabolic requirements, such as hummingbirds, are thus especially vulnerable to the pathogen. Hummingbirds have a strong presence in California, especially in urban environments. However, little is understood regarding the impact of pox virus on hummingbird populations. Currently, diagnosing a pox infection relies on obtaining a tissue biopsy, which poses significant risks to birds and challenges in the field. Understanding the ecology of hummingbird pox viral infections could be advanced by a minimally invasive ante-mortem diagnostic method. Our aim was to address whether pox infections can be diagnosed using integumentary system samples besides tissue biopsies. To meet this goal, we tested multiple integumentary sample types using a quantitative real-time PCR assay. A secondary study goal was to determine which sample types (ranging from minimally to highly invasive sampling) were optimal for identifying infected birds. METHODOLOGY AND PRINCIPAL FINDINGS: Pox-like lesion tissue, pectoral muscle, feathers, toenail clippings, blood, and swabs (both pox-like lesion tissue and non pox-like lesion tissue) were taken from live birds and carcasses of two species of hummingbirds found in California. To maximize successful diagnosis, especially for samples with low viral load, a real-time quantitative PCR assay was developed for detecting the hummingbird-specific Avipoxvirus 4b core protein gene. Avipoxvirus DNA was successfully amplified from all sample types obtained from 27 individuals. These results were compared to those of conventional PCR and comparisons were also made among sample types, utilizing lesion tissue samples as the gold standard. CONCLUSIONS AND SIGNIFICANCE: Hummingbird avian pox can be diagnosed without relying on tissue biopsies. We identify that feather samples, of which contour feathers yielded the best results, can be used for diagnosing infected birds, thus reducing sampling risk. In sum, the real-time PCR assay detected viral DNA in various integumentary system sample types and will be useful in future studies of hummingbird disease ecology.

Anti-microbial activity of whole blood and plasma collected from Anna's Hummingbirds (Calypte anna) against three different microbes.

Hummingbirds are essential pollinators in many ecosystems, making their conservation critical. As is the case with many species, hummingbirds are now facing a variety of challenges resulting from anthropogenic changes. As populations shift and species interactions change, disease is likely to pose a significant threat. There is a basic understanding of which pathogens currently affect a variety of hummingbird species, however there is a paucity of information about their immune systems capacity to kill pathogens and what specific factors may affect immunity. The objective of this study was to gain a basic understanding of the effect of age, sex, and molt on the constitutive innate immunity of hummingbirds. An in vitro assay was used to assess the microbiocidal capacity of the whole blood of Anna's Hummingbirds (Calypte anna) against three different microbes: Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans). The effect of age, sex and molt on anti-microbial capacity varied based on the microbe type. After-hatch-year birds tended to have better anti-microbial capacity compared to hatch-year birds. Male birds had higher anti-microbial activity than female birds, although this was not observed against C. albicans. Molting birds had a weaker antimicrobial activity against E. coli and S. aureus than birds that were not molting. These results represent an important first step towards defining the parameters of constitutive innate immunity of Anna's Hummingbirds as well as providing important knowledge about factors that should be considered when evaluating the health of wild populations.

Correction: Use of RFID technology to characterize feeder visitations and contact network of hummingbirds in urban habitats.

[This corrects the article DOI: 10.1371/journal.pone.0208057.].

Use of RFID technology to characterize feeder visitations and contact network of hummingbirds in urban habitats.

Despite the popular use of hummingbird feeders, there are limited studies evaluating the effects of congregation, sharing food resources and increased contact when hummingbirds visit feeders in urban landscapes. To evaluate behavioral interactions occurring at feeders, we tagged 230 individuals of two species, Anna's and Allen's Hummingbirds, with passive integrated transponder tags and recorded their visits with RFID transceivers at feeders. For detecting the presence of tagged birds, we developed an RFID equipped feeding station using a commercially available antenna and RFID transceiver. Data recorded included the number of feeder visits, time spent at the feeder, simultaneous feeder visitation by different individuals, and identifying which feeders were most commonly visited by tagged birds. For the study period (September 2016 to March 2018), 118,017 detections were recorded at seven feeding stations located at three California sites. The rate of tagged birds returning to RFID equipped feeders at least once was 61.3% (141/230 birds). Females stayed at feeders longer than males per visit. We identified primary, secondary and tertiary feeders at Sites 2 and 3, according to the frequency of visitation to them, with a mean percentage of 86.9% (SD±19.13) visits to a primary feeder for each tagged hummingbird. During spring and summer, hummingbirds visited feeders most often in morning and evening hours. Feeder visits by males overlapped in time with other males more frequently than other females. The analysis of the contact network at the feeders did not distinguish any significant differences between age or sex. Although most hummingbirds visited the feeders during the daytime, our system recorded night feeder visitations (n = 7 hummingbirds) at one site. This efficient use of RFID technology to characterize feeder visitations and contact networks of hummingbirds in urban habitats could be used in the future to elucidate behaviors, population dynamics and community structure of hummingbirds visiting feeders.