A Review of Chlamydial Infections in Wild Birds.

The Chlamydia are a globally distributed genus of bacteria that can infect and cause disease in a range of hosts. Birds are the primary host for multiple chlamydial species. The most well-known of these is Chlamydia psittaci, a zoonotic bacterium that has been identified in a range of wild and domesticated birds. Wild birds are often proposed as a reservoir of Chlamydia psittaci and potentially other chlamydial species. The aim of this review is to present the current knowledge of chlamydial infections in wild avian populations. We focus on C. psittaci but also consider other Chlamydiaceae and Chlamydia-related bacteria that have been identified in wild birds. We summarise the diversity, host range, and clinical signs of infection in wild birds and consider the potential implications of these infections for zoonotic transmission and avian conservation. Chlamydial bacteria have been found in more than 70 species of wild birds, with the greatest chlamydial diversity identified in Europe. The Corvidae and Accipitridae families are emerging as significant chlamydial hosts, in addition to established wild hosts such as the Columbidae. Clarifying the effects of these bacteria on avian host fitness and the zoonotic potential of emerging Chlamydiales will help us to understand the implications of these infections for avian and human health.

Ability to detect antibodies to beak and feather disease virus in blood on filter paper decreases with duration of storage.

BACKGROUND: Beak and feather disease virus (BFDV) is a circovirus that infects captive and wild psittacine birds, and is of conservation concern. The haemagglutination inhibition (HI) assay is used to determine antibody titres against BFDV, and the use of dried blood spots (DBS) on filter paper stored at room temperature has been suggested to be an equally valid technique to the use of frozen serum. However, research on other pathogens has found variable results when investigating the longevity of antibodies stored on DBS at room temperature. Consequently, we aimed to test the temporal stability of antibodies to BFDV in DBS samples stored long-term at room temperature. A further goal was to add to the current knowledge of antibody response to naturally acquired BFDV infection in crimson rosellas (Platycercus elegans). METHODS: Blood was collected from wild P. elegans in Victoria, Australia, that had been live-trapped (n = 9) or necropsied (n = 11). BFDV virus load data were obtained from blood stored in ethanol by real-time quantitative PCR (qPCR); antibody titres were obtained by HI assay from either DBS or serum samples, which had been collected concurrently. All HI assays were performed commercially by the Veterinary Diagnostic Laboratory (VDL) in Charles Sturt University, Australia, who were blind to BFDV blood status. RESULTS: HI titres from DBS stored at room temperature declined significantly over time (~80 weeks). By contrast, frozen serum samples assayed after 80 weeks in storage all had high HI titres, only varying up to one dilution step from the initial HI titres obtained from DBS at 3-6 weeks after sampling. Weak HI titres from DBS samples all came back negative when the test was repeated only nine weeks later. Novel high HI titres were reported in P. elegans, and while most birds with high antibody titres had corresponding negative qPCR results, a single subadult presented with high HI titres and virus load simultaneously. CONCLUSION: Detection of antibodies on filter paper stored at room temperature decreases over time, increasing the chances of false negatives in these samples, and in repeated testing of samples with weak HI titres. Consequently, serum should be the preferred sample type to use for seroepidemiological studies on BFDV in parrots and other bird species. When not possible, it may help to store DBS on filter paper at -20 °C or lower. However, prompt testing of DBS samples (e.g., <6 weeks in storage) is recommended pending further research on antibody temporal stability. We also show that P. elegans, especially adults, can produce high antibody titres against BFDV, which may help them resist infection.

Chlamydial diversity and predictors of infection in a wild Australian parrot, the Crimson Rosella (Platycercus elegans).

Members of the Chlamydia genus are known to cause disease in both humans and animals. A variety of other species in the order Chlamydiales are increasingly being discovered and emerging as potential pathogens, yet there are scarce data on the diversity, prevalence and impacts of these pathogens in wild birds. To address this gap, we investigated which Chlamydiales species are present in a wild population of a common Australian parrot, the Crimson Rosella (Platycercus elegans). We collected cloacal swabs and serum from 136 individuals in south-eastern Australia, over two years, and tested several predictors of prevalence: age, sex, season and breeding status. We used multiple PCR assays to determine bacterial prevalence in cloacal swabs and a solid-phase ELISA to determine seroprevalence. We found Chlamydiales PCR prevalence of 27.7% (95% CI 20.2, 36.2) and identified at least two families (Chlamydiaceae and Parachlamydiaceae). Regarding known chlamydial avian pathogens, we found C. psittaci at 6.2% (95% CI 2.7, 11.8) and C. gallinacea at 4.6% (95% CI 1.7, 9.8) prevalence. We also identified at least two potentially novel Chlamydiales species, of unknown pathogenicity. Sex and breeding status predicted Chlamydiales PCR prevalence, with females more likely to be infected than males, and non-breeding birds more likely to be infected than breeding birds. Seroprevalence was 16% (95% CI 8.8, 25.9). Season and breeding status were strong predictors of seroprevalence, with highest seroprevalence in autumn and in non-breeding birds. Our results reveal a diversity of Chlamydiales species in this abundant wild host, and indicate that host-specific and temporal factors are associated with infection risk. Our findings suggest that wild parrots are a reservoir of both known and novel Chlamydiales lineages, of zoonotic and pathogenic potential.

Species, sex and geographic variation in chlamydial prevalence in abundant wild Australian parrots.

Chlamydia psittaci (order: Chlamydiales) is a globally distributed zoonotic bacterium that can cause potentially fatal disease in birds and humans. Parrots are a major host, yet prevalence and risk factors for infection in wild parrots are largely unknown. Additionally, recent research suggests there is a diverse range of novel Chlamydiales circulating in wildlife. We therefore sampled seven abundant parrot species in south-eastern Australia, taking cloacal swabs and serum from n = 132 wild adults. We determined C. psittaci and Chlamydiales prevalence and seroprevalence, and tested for host species, sex, geographical and seasonal differences, and temporal changes in individual infection status. Across all species, Chlamydiales prevalence was 39.8% (95% CI 31.6, 48.7), C. psittaci prevalence was 9.8% (95% CI 5.7, 16.3) and C. gallinacea prevalence was 0.8% (95% CI 0.1, 4.5). Other Chlamydiales species were not identified to species level. We identified two C. psittaci strains within the 6BC clade, which is highly virulent in humans. Seroprevalence was 37.0% (95% CI 28.5, 46.4). Host species (including crimson rosellas, galahs, sulphur-crested cockatoos and blue-winged parrots) differed in seroprevalence and Chlamydiales prevalence. Galahs had both highest Chlamydiales prevalence (54.8%) and seroprevalence (74.1%). Seroprevalence differed between sites, with a larger difference in males (range 20-63%) than females (29-44%). We reveal a higher chlamydial prevalence than previously reported in many wild parrots, with implications for potential reservoirs, and transmission risks to humans and other avian hosts.

Senescence of song revealed by a long-term study of the Seychelles warbler (Acrocephalus sechellensis).

Senescence is widespread in nature, often resulting in diminishing survival or reproduction with age, but its role in age-dependent variation in sexual traits is often poorly understood. One reason is that few studies of sexual traits consider non-linear relationships with age, or only consider a narrow range of years relative to the life span of the species. Birdsong has evolved to allow assessment of conspecific quality in numerous bird species. Whilst theory and empirical work suggests that song may become more elaborate with age, there are a paucity of long-term studies testing whether song is associated with age or longevity. In particular, the occurrence of song senescence has rarely been demonstrated. Using an exceptional long-term dataset for the Seychelles warbler (Acrocephalus sechellensis), we analysed relationships between male song, age, survival, and longevity. This species is a long-lived songbird with early life increases, followed by senescent declines, in survival and reproduction. The study population (Cousin Island, Seychelles) is a closed population, with no depredation of adults, providing an excellent opportunity to study senescence in free-living animals. We tested whether song traits were related to age at recording, future survival, longevity, and territory quality. We found age-dependent changes in five song traits (duration, maximum frequency, peak frequency of songs, and duration and frequency bandwidth of trills). Relationships with age were quadratic, indicating reversal in the expression of song coinciding with the onset of senescence in reproduction and survival in this species. One song trait (trill bandwidth) had a quadratic relationship with future survival, but no song traits were related to longevity, suggesting age-related patterns were not the result of selective disappearance. Our study provides one of the first examples of functional senescence in song, offering new insights into avian senescence. Late-life declines in avian song, and possibly other sexual traits, may be more common than currently known, and may play a fundamental role in age-dependent changes in reproductive success.

Beak and feather disease virus (BFDV) prevalence, load and excretion in seven species of wild caught common Australian parrots.

Pathogens pose a major risk to wild host populations, especially in the face of ongoing biodiversity declines. Beak and feather disease virus (BFDV) can affect most if not all members of one of the largest and most threatened bird orders world-wide, the Psittaciformes. Signs of disease can be severe and mortality rates high. Its broad host range makes it a risk to threatened species in particular, because infection can occur via spill-over from abundant hosts. Despite these risks, surveillance of BFDV in locally abundant wild host species has been lacking. We used qPCR and haemagglutination assays to investigate BFDV prevalence, load and shedding in seven abundant host species in the wild in south-east Australia: Crimson Rosellas (Platycercus elegans), Eastern Rosellas (Platycercus eximius), Galahs (Eolophus roseicapillus), Sulphur-crested Cockatoos (Cacatua galerita), Blue-winged Parrots (Neophema chrysostoma), Rainbow Lorikeets (Trichoglossus moluccanus) and Red-rumped Parrots (Psephotus haematonotus). We found BFDV infection in clinically normal birds in six of the seven species sampled. We focused our analysis on the four most commonly caught species, namely Crimson Rosellas (BFDV prevalence in blood samples: 41.8%), Sulphur-crested Cockatoos (20.0%), Blue-winged Parrots (11.8%) and Galahs (8.8%). Species, but not sex, was a significant predictor for BFDV prevalence and load. 56.1% of BFDV positive individuals were excreting BFDV antigen into their feathers, indicative of active viral replication with shedding. Being BFDV positive in blood samples predicted shedding in Crimson Rosellas. Our study confirms that BFDV is endemic in our study region, and can inform targeted disease management by providing comparative data on interspecies variation in virus prevalence, load and shedding.

A non-invasive method to assess environmental contamination with avian pathogens: beak and feather disease virus (BFDV) detection in nest boxes.

Indirect transmission of pathogens can pose major risks to wildlife, yet the presence and persistence of wildlife pathogens in the environment has been little studied. Beak and feather disease virus (BFDV) is of global conservation concern: it can infect all members of the Psittaciformes, one of the most threatened bird orders, with infection often being lethal. Indirect transmission of BFDV through contaminated nest hollows has been proposed as a major infection source. However, data on whether and for how long nest sites in the wild remain contaminated have been absent. We determined the BFDV status of birds (parents and nestlings) for 82 nests of Crimson Rosellas, Platycercus elegans and Eastern Rosellas, Platycercus eximius. In 11 of these nests (13.4%, 95% confidence interval 6.9-22.7), we found an infected parent or nestling. Using nest swabs, we then compared BFDV presence at three points in time (before, during and after breeding) in three groups of nest boxes. These were nest boxes occupied by infected birds, and two control groups (nest boxes occupied by uninfected birds, and unoccupied nest boxes). Detection of BFDV on nest swabs was strongly associated with the infection status of parents in each nest box and with the timing of breeding. During breeding, boxes occupied by BFDV-positive birds were significantly more likely to have BFDV-positive nest swabs than boxes occupied by BFDV-negative birds; nest swabs tested BFDV-positive in 80% (28.4-99.5) of nests with parental antigen excretion, 66.7% (9.4-99.2) of nests occupied by parents with BFDV-positive cloacal swabs and 66.7% (22.3-95.7) of nests occupied by parents with BFDV-positive blood. 0% (0-52.2) of nests with BFDV-positive nestlings had BFDV-positive nest swabs. Across all boxes occupied by BFDV-positive birds (parents or nestlings), no nest swabs were BFDV-positive before breeding, 36.4% (95% CI 10.9-69.2) were positive during breeding and 9.1% (0.2-41.3) remained positive after breeding. BFDV was present on nest swabs for up to 3.7 months. Our study provides novel insights into the potential role of nest cavities and other fomites in indirect transmission of BFDV, and possibly other pathogens, and offers a non-invasive method for surveillance of pathogens in wild bird populations.

Seasonal fluctuation of beak and feather disease virus (BFDV) infection in wild Crimson Rosellas (Platycercus elegans).

Understanding patterns of pathogen emergence can help identify mechanisms involved in transmission dynamics. Beak and feather disease virus (BFDV) poses a major threat world-wide to wild and captive parrots. Yet data from wild birds on seasonal fluctuations in prevalence and infection intensity, and thereby the potential high-risk times for virus transmission, have been lacking. We screened wild Crimson Rosellas (Platycercus elegans) for BFDV in blood and cloacal swabs. Prevalence in blood samples and cloacal swabs, as well as viral load varied with Julian date and in blood, were highest after the breeding season. Breeding birds had lower viral load and lower BFDV prevalence in blood than non-breeding birds (10.1% prevalence in breeding vs. 43.2% in non-breeding birds). BFDV prevalence was much higher in younger (<3 years) than older (≥3 years) birds for both blood samples (42.9% vs. 4.5%) and cloacal swabs (56.4% vs. 12.3%). BFDV status in blood and cloacal samples was not correlated within individuals. We show that, at least in P. elegans, BFDV infection seems to occur year-round, with seasonal changes in prevalence and load found in our samples. Our analyses suggest that the seasonal changes were associated primarily with the breeding season. We also discuss age and sex as important predictors of BFDV infection.

Do glucocorticoids or carotenoids mediate plumage coloration in parrots? An experiment in Platycercus elegans.

Conspicuous coloration can indicate phenotypic quality, and may reflect exposure or vulnerability to stress, or access to essential nutrients such as pigments. Although the production of pigmented colours is well understood, much less is known about how structural colours are affected by physiological state. In this study, we tested whether glucocorticoids (corticosterone) predicted expression of plumage coloration in an Australian parrot, the crimson rosella (Platycercus elegans). Parrots provide an interesting and unique test, as they possess conspicuous coloration produced by distinctive pigments known as psittacofulvins, in addition to structural coloration. We have previously documented that coloration in P. elegans is condition-dependent and responds to dietary manipulation. Here, n = 21 P. elegans underwent a dietary manipulation (including food restriction or carotenoid supplementation) during which they moulted, and the change in reflectance was measured for three structural and three pigmentary plumage patches. Stress-induced corticosterone (10 min after handling) measured at the start of the experiment predicted change in coloration in two pigmentary patches (crown and front). We also found that change in stress-induced corticosterone during the experiment was associated with the change in coloration of the crown and two structural patches (cheek and epaulette). Baseline corticosterone (<3 min after handling) was not associated with any measure of coloration. We found no effects of dietary manipulation on baseline or stress-induced corticosterone, but carotenoid supplementation was associated with an increase in a measure of chronic stress (heterophil/lymphocyte ratio), and the corticosterone response to handling decreased over the course of the study. Our results suggest that corticosterone may be linked to colour expression more broadly than previously recognised, including psittacofulvin and structural coloration in parrots, and they confirm the independence of plumage pigmentation in parrots from carotenoid accumulation. Moreover, our study provides new insight into the stress responses of Psittaciformes, one of the most highly threatened avian orders.

Host heterozygosity and genotype rarity affect viral dynamics in an avian subspecies complex.

Genetic diversity at community, population and individual levels is thought to influence the spread of infectious disease. At the individual level, inbreeding and heterozygosity are associated with increased risk of infection and disease severity. Host genotype rarity may also reduce infection risk if pathogens are co-adapted to common or local hosts, but to date, no studies have investigated the relative importance of genotype rarity and heterozygosity for infection in a wild, sexually reproducing vertebrate. With beak and feather disease virus (BFDV) infection in a wild parrot (Platycercus elegans), we show that both heterozygosity and genotype rarity of individual hosts predicted infection, but in contrasting ways. Heterozygosity was negatively associated with probability of infection, but not with infection load. In contrast, increased host genotype rarity was associated with lower viral load in infected individuals, but did not predict infection probability. These effects were largely consistent across subspecies, but were not evident at the population level. Subspecies and age were also strongly associated with infection. Our study provides novel insights into infection dynamics by quantifying rarity and diversity simultaneously. We elucidate roles that host genetic diversity can play in infection dynamics, with implications for understanding population divergence, intraspecific diversity and conservation.

Intraspecific geographic variation in rod and cone visual pigment sensitivity of a parrot, Platycercus elegans.

Variation in wavelength sensitivity among subspecies is unknown among vertebrates. The parrot Platycercus elegans has extreme plumage variation between subspecies ranging from pale yellow to crimson which, with differences in background colour and light environment between subspecies, makes it a good candidate for the evolution of within-species differences in vision. We report differences in visual pigments between populations of P. elegans from two subspecies, providing the first known support for population and subspecies variation in visual pigments within a vertebrate species; it is also the first instance of intraspecific variation in rod sensitivity within any vertebrate species. Differences in wavelength sensitivity of rods and cones corresponded to geographic differences in plumage colour. Between study populations, visual pigments varied but not oil droplets. Adaptive functions for the visual pigment differences are untested but they could cause divergence in behaviours associated with colour as well as in dim light, and provide insights into the role of senses in divergence and speciation.

Phylogenetic analysis of beak and feather disease virus across a host ring-species complex.

Pathogens have been hypothesized to play a major role in host diversity and speciation. Susceptibility of hybrid hosts to pathogens is thought to be a common phenomenon that could promote host population divergence and subsequently speciation. However, few studies have tested for pathogen infection across animal hybrid zones while testing for codivergence of the pathogens in the hybridizing host complex. Over 8 y, we studied natural infection by a rapidly evolving single-strand DNA virus, beak and feather diseases virus (BFDV), which infects parrots, exploiting a host-ring species complex (Platycercus elegans) in Australia. We found that host subspecies and their hybrids varied strikingly in both BFDV prevalence and load: both hybrid and phenotypically intermediate subspecies had lower prevalence and load compared with parental subspecies, while controlling for host age, sex, longitude and latitude, as well as temporal effects. We sequenced viral isolates throughout the range, which revealed patterns of genomic variation analogous to Mayr's ring-species hypothesis, to our knowledge for the first time in any host-pathogen system. Viral phylogeny, geographic location, intraspecific host density, and parrot community diversity and composition did not explain the differences in BFDV prevalence or load between subpopulations. Overall, our analyses suggest that functional host responses to infection, or force of infection, differ between subspecies and hybrids. Our findings highlight the role of host hybridization and clines in altering host-pathogen interactions, dynamics that can have important implications for models of speciation with gene flow, and offer insights into how pathogens may adapt to diverging host populations.

How parrots see their colours: novelty in the visual pigments of Platycercus elegans.

Intraspecific differences in retinal physiology have been demonstrated in several vertebrate taxa and are often subject to adaptive evolution. Nonetheless, such differences are currently unknown in birds, despite variations in habitat, behaviour and visual stimuli that might influence spectral sensitivity. The parrot Platycercus elegans is a species complex with extreme plumage colour differences between (and sometimes within) subspecies, making it an ideal candidate for intraspecific differences in spectral sensitivity. Here, the visual pigments of P. elegans were fully characterised through molecular sequencing of five visual opsin genes and measurement of their absorbance spectra using microspectrophotometry. Three of the genes, LWS, SW1 and SWS2, encode for proteins similar to those found in other birds; however, both the RH1 and RH2 pigments had polypeptides with carboxyl termini of different lengths and unusual properties that are unknown previously for any vertebrate visual pigment. Specifically, multiple RH2 transcripts and protein variants (short, medium and long) were identified for the first time that are generated by alternative splicing of downstream coding and non-coding exons. Our work provides the first complete characterisation of the visual pigments of a parrot, perhaps the most colourful order of birds, and moreover suggests more variability in avian eyes than hitherto considered.

Learned vocal variation is associated with abrupt cryptic genetic change in a parrot species complex.

Contact zones between subspecies or closely related species offer valuable insights into speciation processes. A typical feature of such zones is the presence of clinal variation in multiple traits. The nature of these traits and the concordance among clines are expected to influence whether and how quickly speciation will proceed. Learned signals, such as vocalizations in species having vocal learning (e.g. humans, many birds, bats and cetaceans), can exhibit rapid change and may accelerate reproductive isolation between populations. Therefore, particularly strong concordance among clines in learned signals and population genetic structure may be expected, even among continuous populations in the early stages of speciation. However, empirical evidence for this pattern is often limited because differences in vocalisations between populations are driven by habitat differences or have evolved in allopatry. We tested for this pattern in a unique system where we may be able to separate effects of habitat and evolutionary history. We studied geographic variation in the vocalizations of the crimson rosella (Platycercus elegans) parrot species complex. Parrots are well known for their life-long vocal learning and cognitive abilities. We analysed contact calls across a ca 1300 km transect encompassing populations that differed in neutral genetic markers and plumage colour. We found steep clinal changes in two acoustic variables (fundamental frequency and peak frequency position). The positions of the two clines in vocal traits were concordant with a steep cline in microsatellite-based genetic variation, but were discordant with the steep clines in mtDNA, plumage and habitat. Our study provides new evidence that vocal variation, in a species with vocal learning, can coincide with areas of restricted gene flow across geographically continuous populations. Our results suggest that traits that evolve culturally can be strongly associated with reduced gene flow between populations, and therefore may promote speciation, even in the absence of other barriers.

Absorbance of retinal oil droplets of the budgerigar: sex, spatial and plumage morph-related variation.

Intraspecific variation in photoreceptor physiology is known in several vertebrate taxa, but is currently unknown in birds, despite many avian traits varying intraspecifically, and avian visual ecology encompassing a wide range of environments and visual stimuli, which might influence spectral sensitivity. Avian retinal photoreceptors contain light absorbing carotenoid-rich oil droplets that affect vision. Carotenoids are also important plumage components. However, our understanding of the regulation of carotenoids in oil droplets remains rudimentary. Among birds, Melopsittacus undulatus has probably the best-studied colour vision, shows profound intraspecific variation in plumage colour, and increased plasma carotenoids during moult. We used microspectrophotometry to determine whether a relationship exists between oil droplet carotenoid concentration and plumage pigmentation, and tested for sex and spatial variation in droplet absorbance across the retina. Absorbance of one variety of P-type droplets was higher in males. No relationship was found between droplet absorbance and plumage colour. We found a spatial pattern of droplets absorbance across the retina that matched a pattern found in another parrot, and other avian species. Our work provides insights into the development and maintenance of retinal oil droplets and suggests a common mechanism and function for carotenoid deposition in the retina across bird species.

Ultraviolet-sensitive vision in long-lived birds.

Long-term exposure to ultraviolet (UV) light generates substantial damage, and in mammals, visual sensitivity to UV is restricted to short-lived diurnal rodents and certain marsupials. In humans, the cornea and lens absorb all UV-A and most of the terrestrial UV-B radiation, preventing the reactive and damaging shorter wavelengths from reaching the retina. This is not the case in certain species of long-lived diurnal birds, which possess UV-sensitive (UVS) visual pigments, maximally sensitive below 400 nm. The Order Psittaciformes contains some of the longest lived bird species, and the two species examined so far have been shown to possess UVS pigments. The objective of this study was to investigate the prevalence of UVS pigments across long-lived parrots, macaws and cockatoos, and therefore assess whether they need to cope with the accumulated effects of exposure to UV-A and UV-B over a long period of time. Sequences from the SWS1 opsin gene revealed that all 14 species investigated possess a key substitution that has been shown to determine a UVS pigment. Furthermore, in vitro regeneration data, and lens transparency, corroborate the molecular findings of UV sensitivity. Our findings thus support the claim that the Psittaciformes are the only avian Order in which UVS pigments are ubiquitous, and indicate that these long-lived birds have UV sensitivity, despite the risks of photodamage.

Avian retinal oil droplets: dietary manipulation of colour vision?

Avian vision is highly developed, with bird retinas containing rod and double-cone photoreceptors, plus four classes of single cones subserving tetrachromatic colour vision. Cones contain an oil droplet, rich in carotenoid pigments (except VS/ultraviolet-sensitive cones), that acts as a filter, substantially modifying light detected by the photoreceptor. Using dietary manipulations, we tested the effects of carotenoid availability on oil droplet absorbance properties in two species: Platycercus elegans and Taeniopygia guttata. Using microspectrophotometry, we determined whether manipulations affected oil droplet carotenoid concentration and whether changes would alter colour discrimination ability. In both species, increases in carotenoid concentration were found in carotenoid-supplemented birds, but only in the double cones. Magnitudes of effects of manipulations were often dependent on retinal location. The study provides, to our knowledge, the first experimental evidence of dietary intake over a short time period affecting carotenoid concentration of retinal oil droplets. Moreover, the allocation of carotenoids to the retina by both species is such that the change potentially preserves the spectral tuning of colour vision. Our study generates new insights into retinal regulation of carotenoid concentration of oil droplets, an area about which very little is known, with implications for our understanding of trade-offs in carotenoid allocation in birds.

Where and when does a ring start and end? Testing the ring-species hypothesis in a species complex of Australian parrots.

Speciation, despite ongoing gene flow can be studied directly in nature in ring species that comprise two reproductively isolated populations connected by a chain or ring of intergrading populations. We applied three tiers of spatio-temporal analysis (phylogeny/historical biogeography, phylogeography and landscape/population genetics) to the data from mitochondrial and nuclear genomes of eastern Australian parrots of the Crimson Rosella Platycercus elegans complex to understand the history and present genetic structure of the ring they have long been considered to form. A ring speciation hypothesis does not explain the patterns we have observed in our data (e.g. multiple genetic discontinuities, discordance in genotypic and phenotypic assignments where terminal differentiates meet). However, we cannot reject that a continuous circular distribution has been involved in the group's history or indeed that one was formed through secondary contact at the 'ring's' east and west; however, we reject a simple ring-species hypothesis as traditionally applied, with secondary contact only at its east. We discuss alternative models involving historical allopatry of populations. We suggest that population expansion shown by population genetics parameters in one of these isolates was accompanied by geographical range expansion, secondary contact and hybridization on the eastern and western sides of the ring. Pleistocene landscape and sea-level and habitat changes then established the birds' current distributions and range disjunctions. Populations now show idiosyncratic patterns of selection and drift. We suggest that selection and drift now drive evolution in different populations within what has been considered the ring.