Improving provenance studies in migratory birds when using feather hydrogen stable isotopes

Hydrogen stable isotopes (δ²H) in feathers are used to determine the origin and migration strategy of birds. To identify the geographic location of the site of feather synthesis, calibration curves for the relation between feather δ²H and amount‐weighted growing‐season δ²H in precipitation are used to generate feather δ²H isoscapes. Factors like species, age and year might generate isotopic variation in calibration curves, but the extent to which accounting for variation may improve calibration curves and hence provenance determination of birds, is unknown. We compared three European calibration curves: 1) an existing multi‐species curve, uncorrected for age and year variation, and two species‐specific calibration curves, based on mallard Anas platyrhynchos feathers, of 2) varying age and year, and 3) juvenile natal origin, corrected for year variation. Calibration curves using ordinary least square linear regression (OLS) as opposed to standard major axis regression showed least bias in estimation. As expected, we found that a single species (mallard) OLS calibration curve corrected for age and year yielded the highest coefficient of determination, but was still surprisingly similar to the other two calibration curves. Nevertheless, when using feathers of known‐origin to assess provenance accuracy, the calibration curve that accounted for species, age and year variation yielded the best prediction in as many as 59% of the cases. Our study is the first to demonstrate implications of isotopic variation on assessing the origin of individual birds, but also highlights the relatively small gain in precision that is achieved by generating species, age and year specific calibration curves rather than resorting to more general alternatives.     

Using stable hydrogen and oxygen isotope measurements of feathers to infer geographical origins of migrating European birds

Successful application of stable-hydrogen isotope measurements (D_f) of feathers to track origins of migratory birds and other wildlife requires a fundamental understanding of the correlation between D_f and deuterium patterns in rainfall (D_p) over continental scales. A strong correlation between D_p and D_f has been confirmed for birds and insects in North America, but not yet for other continents. Here, we compare D_f data from resident European birds to new D_p basemaps for Europe. Three maps, representing growing-season and mean annual D_p estimates from an elevation-explicit, detrended interpolation model and growing-season D_p estimates from simple Kriging, all indicate that strong isotope gradients occur across Europe with a general depletion occurring in a northeast direction. The feather data, representing 141 individuals of 25 avian species from 38 sites, ranged from –131 to –38. Regression analysis showed that strong correlations existed between both mean annual and growing-season D_p estimated by detrended interpolation and D_f of non-aquatic and non-corvid birds (r²=0.66 and 0.65, respectively). We also examined mean annual and growing-season ¹⁸O_p vs. ¹⁸O_f for our samples. Both oxygen regressions were similar (r²=0.56 and 0.57, respectively) but poorer than for deuterium. Our study reveals that D measurements of feathers from migratory birds in Europe may be used to track their origin and movements, and so provide a powerful investigative tool for avian migration research in Europe.     

Diffuse migratory connectivity in two species of shrubland birds: evidence from stable isotopes

Connecting seasonal ranges of migratory birds is important for understanding the annual template of stressors that influence their populations. Brewer’s sparrows (Spizella breweri) and sagebrush sparrows (Artemisiospiza nevadensis) share similar sagebrush (Artemisia spp.) habitats for breeding but have different population trends that might be related to winter location. To link breeding and winter ranges, we created isoscapes of deuterium [stable isotope ratio (δ) of deuterium; δ ²H] and nitrogen (δ ¹⁵N) for each species modeled from isotope ratios measured in feathers of 264 Brewer’s and 82 sagebrush sparrows and environmental characteristics at capture locations across their breeding range. We then used feather $\delta^{2} {\text{H}}_{\text{f}}$ and $\delta^{15} {\text{N}}_{\text{f}}$ measured in 1,029 Brewer’s and 527 sagebrush sparrows captured on winter locations in southwestern United States to assign probable breeding ranges. Intraspecies population mixing from across the breeding range was strong for both Brewer’s and sagebrush sparrows on winter ranges. Brewer’s sparrows but not sagebrush sparrows were linked to more northerly breeding locations in the eastern part of their winter range. Winter location was not related to breeding population trends estimated from US Geological Survey Breeding Bird Survey routes for either Brewer’s or sagebrush sparrows. Primary drivers of population dynamics are likely independent for each species; Brewer’s and sagebrush sparrows captured at the same winter location did not share predicted breeding locations or population trends. The diffuse migratory connectivity displayed by Brewer’s and sagebrush sparrows measured at the coarse spatial resolution in our analysis also suggests that local environments rather than broad regional characteristics are primary drivers of annual population trends.     

The origins of northern European Autographa gamma individuals evaluated using hydrogen stable isotopes

1. Many insect species are migratory. As the spring progresses, adults gradually depart from their over‐wintering habitats and arrive in northern zones where they reproduce during the summer. Understanding this transgenerational and highly adaptive migratory behaviour is crucial when interpreting life cycle dynamics of many insect pests. 2. Origins of migratory silver Y moths, Autographa gamma (Linnaeus, 1758), captured from Finland, were studied with stable hydrogen analysis of their wings (δ²H_w). 3. The difference between spring and autumn generation δ²H_w values indicate different geographical natal origins. The probability surface map shows that the spring generation probably emerged in central Europe (Benelux countries, Germany and parts of France). 4. A negative correlation between the δ²H_w values and the migrants' capture year suggests that a warming climate may have driven the transgenerational migratory stages northwards during the last century.     

Ancient origins and multiple appearances of carotenoid-pigmented feathers in birds

The broad palette of feather colours displayed by birds serves diverse biological functions, including communication and camouflage. Fossil feathers provide evidence that some avian colours, like black and brown melanins, have existed for at least 160 million years (Myr), but no traces of bright carotenoid pigments in ancient feathers have been reported. Insight into the evolutionary history of plumage carotenoids may instead be gained from living species. We visually surveyed modern birds for carotenoid-consistent plumage colours (present in 2956 of 9993 species). We then used high-performance liquid chromatography and Raman spectroscopy to chemically assess the family-level distribution of plumage carotenoids, confirming their presence in 95 of 236 extant bird families (only 36 family-level occurrences had been confirmed previously). Using our data for all modern birds, we modelled the evolutionary history of carotenoid-consistent plumage colours on recent supertrees. Results support multiple independent origins of carotenoid plumage pigmentation in 13 orders, including six orders without previous reports of plumage carotenoids. Based on time calibrations from the supertree, the number of avian families displaying plumage carotenoids increased throughout the Cenozoic, and most plumage carotenoid originations occurred after the Miocene Epoch (23 Myr). The earliest origination of plumage carotenoids was reconstructed within Passeriformes, during the Palaeocene Epoch (66–56 Myr), and not at the base of crown-lineage birds.     

Estimating the timing of diet shifts using stable isotopes

Stable isotope analysis has become an important tool in studies of trophic food webs and animal feeding patterns. When animals undergo rapid dietary shifts due to migration, metamorphosis, or other reasons, the isotopic composition of their tissues begins changing to reflect that of their diet. This can occur both as a result of growth and metabolic turnover of existing tissue. Tissues vary in their rate of isotopic change, with high turnover tissues such as liver changing rapidly, while relatively low turnover tissues such as bone change more slowly. A model is outlined that uses the varying isotopic changes in multiple tissues as a chemical clock to estimate the time elapsed since a diet shift, and the magnitude of the isotopic shift in the tissues at the new equilibrium. This model was tested using published results from controlled feeding experiments on a bird and a mammal. For the model to be effective, the tissues utilized must be sufficiently different in their turnover rates. The model did a reasonable job of estimating elapsed time and equilibrial isotopic changes, except when the time since the diet shift was less than a small fraction of the half-life of the slowest turnover tissue or greater than 5–10 half-lives of the slowest turnover tissue. Sensitivity analyses independently corroborated that model estimates became unstable at extremely short and long sample times due to the effect of random measurement error. Subject to some limitations, the model may be useful for studying the movement and behavior of animals changing isotopic environments, such as anadromous fish, migratory birds, animals undergoing metamorphosis, or animals changing diets because of shifts in food abundance or competitive interactions.     

Tracing origins and migration of wildlife using stable isotopes: a review

To understand the ecology of migratory animals it is important to link geographic regions used by individuals including breeding, wintering, and intermediate stopover sites. Previous conventional approaches used to track animal movements have relied on extrinsic markers and typically the subsequent recovery of individuals. This approach has generally been inappropriate for most small, or non-game animals. The use of intrinsic markers such as fatty acid profiles, molecular DNA analyses, and the measurement of naturally occurring stable isotopes in animal tissues offer alternative approaches. This paper reviews the use of stable isotope analyses (primarily δ¹³C, δ¹⁵N, δ³⁴S, δD, δ⁸⁷Sr) to trace nutritional origin and migration in animals. This approach relies on the fact that foodweb isotopic signatures are reflected in the tissues of organisms and that such signatures can vary spatially based on a variety of biogeochemical processes. Organisms moving between isotopically distinct foodwebs can carry with them information on the location of previous feeding. Such an approach has been used to track animal use of inshore versus offshore, marine versus freshwater, terrestrial C₃ versus marine, terrestrial mesic versus xeric, and C₃ versus C₄ or Crassulacean acid metabolism foodwebs. More recently, the use of stable hydrogen isotope analyses (δD) to link organisms to broad geographic origin in North America is based on large-scale isotopic contours of growing-season average δD values in precipitation. This technique, especially when combined with the assay of other stable isotopes, will be extremely useful in helping to track migration and movement of a wide range of animals from insects to birds and mammals. Future research to refine our understanding of natural and anthropogenic-induced isotopic gradients in nature, and to explore the use of stable isotopes of other elements, is recommended. Received: 1 July 1998 / Accepted: 9 December 1998     

Variability in stable isotopes of snowy owl feathers and contribution of marine resources to their winter diet

The snowy owl is an elusive arctic predator known for its nomadic behaviour. Satellite tracking has revealed that some adult snowy owls could make an extensive use of the marine environment during the non‐breeding season. However, the relative contribution of marine resources to their diet is unknown. Stable isotope analyses can be useful to document the diet of mobile animals during periods of the year when individuals are less accessible. This study aimed to assess variation in isotopic values (δ¹³C and δ¹⁵N) of various feather types, and the usefulness of feathers to determine the contribution of the marine environment to the winter diet of snowy owls captured in summer. We sampled feathers coming from 6 body regions of 18 breeding females at two sites in the eastern Canadian Arctic in 2013 and 2014. Prior to analyses, diet‐tissue discrimination factors of snowy owl feathers were established in captivity. Variability in isotopic values among feather types was relatively low and pairwise correlations in isotopic values between feathers on the same individual were variable and often low, which suggests differences in the diet at the time when various feathers were synthesized. Diet reconstruction models detected a contribution of marine sources to snowy owl feathers ranging from 4 to 19% among feather types. However, the marine contribution was highly variable when single feathers were examined within individuals, ranging from 3 to 71%. This indicated that no single feather type could be used alone to reliably infer the contribution of marine resources to the winter diet of owls, possibly due to a high variability in the timing and sequence of molt. For asynchronous molters like snowy owls, we recommend sampling multiple feathers from various body regions, excluding wing feathers, to investigate winter diet or habitat use.     

Estimating niche width using stable isotopes in the face of habitat variability: a modelling case study in the marine environment

Distributions of stable isotopes have been used to infer an organism's trophic niche width, the 'isotopic niche', and examine resource partitioning. Spatial variation in the isotopic composition of prey may however confound the interpretation of isotopic signatures especially when foragers exploit resources across numerous locations. In this study the isotopic compositions from marine assemblages are modelled to determine the role of variation in the signature of prey items and the effect of dietary breadth and foraging strategies on predator signatures. Outputs from the models reveal that isotopic niche widths can be greater for populations of dietary specialists rather than for generalists, which contravenes what is generally accepted in the literature. When a range of different mixing models are applied to determine if the conversion from δ to p-space can be used to improve model accuracy, predator signature variation is increased rather than model precision. Furthermore the mixing models applied failed to correctly identify dietary specialists and/or to accurately estimate diet contributions that may identify resource partitioning. The results presented illustrate the need to collect sufficiently large sample sizes, in excess of what is collected under most current studies, across the complete distribution of a species and its prey, before attempts to use stable isotopes to make inferences about niche width can be made.     

Determining the sources of calcium for migratory songbirds using stable strontium isotopes

We investigated natural variations in the stable isotopic composition of strontium (a surrogate for calcium) in the bones of a single species of breeding migratory songbird, as well as in their eggshells, egg contents, and food sources. We use this information to determine the sources of calcium to these migratory songbirds and their offspring. Samples were collected from two locations in the northeastern USA (Hubbard Brook, NH, and Downer Forest, VT.) that differed in soil geochemistry. The mean ⁸⁷Sr/⁸⁶Sr ratios of food items (caterpillars and snails), eggshells, and egg contents were indistinguishable within each site, but significantly different between the two sites. Mean ⁸⁷Sr/⁸⁶Sr ratios for the bones of adult females were significantly different between the two sites, but values were significantly lower than those of food items and eggshells at each site. Two of four adult individuals studied at each site had ⁸⁷Sr/⁸⁶Sr ratios lower than the entire range of values for local food sources. Mixing calculations indicate that up to 60% of skeletal strontium and calcium was derived from foods consumed in the winter grounds where lower ⁸⁷Sr/⁸⁶Sr ratios predominate. At each study site, the ⁸⁷Sr/⁸⁶Sr ratio of eggshells differed significantly between clutches, but the mean clutch ⁸⁷Sr/⁸⁶Sr ratios were unrelated to the skeletal ⁸⁷Sr/⁸⁶Sr ratio of the laying adult. These findings suggest that strontium (and hence calcium) for eggshell production in this species is derived predominantly from local food sources in breeding areas. Thus, reductions in available calcium in northern temperate ecosystems due to the influences of acid deposition could be potentially harmful to this and other species of migratory bird.     

Factors that influence assimilation rates and fractionation of nitrogen and carbon stable isotopes in avian blood and feathers

By switching great skuas Catharacta skua from one isotopically distinct diet to another, we measured diet-tissue discrimination factors and tested the assumption that dietary nitrogen and carbon isotope signatures are incorporated into blood and feathers at similar rates. We also examined the effects of metabolic rate and looked for evidence of isotopic routing. We found that blood delta(15)N and delta(13)C signatures altered after the diet switch at similar rates (14.4 d and 15.7 d, respectively). Qualitative analyses imply that the same was true with feathers. Mass balance calculations suggest that only a small amount of lipid is likely to be involved in the synthesis of blood and feathers. Differences in diet-tissue discrimination factors before and after the diet switch may mean that toward the end of the experiment, some of the nutrients for blood synthesis had been coming from stores. Repeated measures mixed models provided evidence that increases in metabolic rate might accelerate fractional turnover rates in blood. There is a need for more laboratory-based experimental isotope studies in order to address further questions that this study has raised.     

Connecting breeding and wintering grounds of Neotropical migrant songbirds using stable hydrogen isotopes: a call for an isotopic atlas of migratory connectivity

There is an overdue and urgent need to establish patterns of migratory connectivity linking breeding grounds, stopover sites, and wintering grounds of migratory birds. Such information allows more effective application of conservation efforts by applying focused actions along movement trajectories at the population level. Stable isotope methods, especially those using stable hydrogen isotope abundance in feathers (δ²H_f) combined with Bayesian assignment techniques incorporating prior information such as relative abundance of breeding birds, now provide a fast and reliable means of establishing migratory connectivity, especially for Neotropical migrants that breed in North America and molt prior to fall migration. Here we demonstrate how opportunistic sampling of feathers of 30 species of wintering birds in Cuba, Venezuela, Guatemala, Puerto Rico, and Mexico, regions that have typically been poorly sampled for estimating migratory connectivity, can be assigned to breeding areas in North America through both advanced spatial assignment to probability surfaces and through simpler map lookup approaches. Incorporating relative abundance information from the North American Breeding Bird Survey in our Bayesian assignment models generally resulted in a reduction in potential assignment areas on breeding grounds. However, additional tools to constrain longitude such as DNA markers or other isotopes would be desirable for establishing breeding or molt origins of species with broad longitudinal distributions. The isotope approach could act as a rapid means of establishing basic patterns of migratory connectivity across numerous species and populations. We propose a large‐scale coordinated sampling effort on the wintering grounds to establish an isotopic atlas of migratory connectivity for North American Neotropical migrants and suggest that isotopic variance be considered as a valuable metric to quantify migratory connectivity. This initiative could then act as a strategic template to guide further efforts involving stable isotopes, light‐sensitive geolocators, and other technologies.     

Estimating the diets of animals using stable isotopes and a comprehensive Bayesian mixing model

Using stable isotope mixing models (SIMMs) as a tool to investigate the foraging ecology of animals is gaining popularity among researchers. As a result, statistical methods are rapidly evolving and numerous models have been produced to estimate the diets of animals--each with their benefits and their limitations. Deciding which SIMM to use is contingent on factors such as the consumer of interest, its food sources, sample size, the familiarity a user has with a particular framework for statistical analysis, or the level of inference the researcher desires to make (e.g., population- or individual-level). In this paper, we provide a review of commonly used SIMM models and describe a comprehensive SIMM that includes all features commonly used in SIMM analysis and two new features. We used data collected in Yosemite National Park to demonstrate IsotopeR's ability to estimate dietary parameters. We then examined the importance of each feature in the model and compared our results to inferences from commonly used SIMMs. IsotopeR's user interface (in R) will provide researchers a user-friendly tool for SIMM analysis. The model is also applicable for use in paleontology, archaeology, and forensic studies as well as estimating pollution inputs.     

Estimating the timing of growth rings in Atlantic cod otoliths using stable oxygen isotopes

A technique involving micro‐scale sampling of otolith carbonate and analyses of stable oxygen isotope composition was used to relate the zone appearance of the otolith to the seasonal temperature cycle. Otolith opacity could then be related to the timing of zone formation. Otoliths from two groups of Atlantic cod Gadus morhua held under known temperature conditions over a period of 4 and 6 years were examined. The otolith translucency followed the same pattern as the estimated temperature (from otolith δ¹⁸O values) in the yearly increments three and four, meaning that the translucent zones were deposited at the seasonal highest temperature in late summer and early autumn. The relative light intensities of otolith yearly increments five and six of older fish (deposited in the same years), however, were not significantly correlated to the estimated temperatures since increased otolith translucency also occurred at low temperatures. This might have been caused by stress in connection with gonad development or starvation during the spawning period. The results showed that this method of coupling otolith opacity and stable oxygen isotope composition can be used to estimate the timing of zone formations in otoliths.     

Microbiological fractionation of stable sulfur isotopes: A review and critique

Microbiological transformations of sulfur compounds discriminate to various degrees between the stable sulfur isotopes ³²S and ³⁴S. Comparatively little is known on isotopic effects associated with sulfur‐oxidizing organisms, and the interpretation of results is complicated since the sulfur pathways are poorly defined and compounds containing two or more sulfur atoms at different oxidation states may be involved. Dissimilatory reduction of sulfate, and sulfite reduction by certain assimilatory microorganisms, causes particularly marked isotopic effects, the expression of which depends on the extent of reaction and other incompletely defined environmental conditions. Models have been proposed to account for these effects based on current knowledge of the reduction pathways. Many of the trends observed during dissimilatory sulfate reduction in the laboratory can also be found in the modern environment leaving little doubt that microbiological factors play a significant role in determining sulfur isotope distributions in nature. However, unusually large isotopic effects, rarely approached in the laboratory, are often observed in nature. The reasons for this are not entirely clear, but in sediments it is possible that diffusional isotopic effects are imposed on biological effects.     

Tracing energy flow in stream food webs using stable isotopes of hydrogen

1. Use of the natural ratios of carbon and nitrogen stable isotopes as tracers of trophic interactions has some clear advantages over alternative methods for food web analyses, yet is limited to situations where organic materials of interest have adequate isotopic separation between potential sources. This constrains the use of natural abundance stable isotope approaches to a subset of ecosystems with biogeochemical conditions favourable to source separation. 2. Recent studies suggest that stable hydrogen isotopes (δD) could provide a robust tracer to distinguish contributions of aquatic and terrestrial production in food webs, but variation in δD of consumers and their organic food sources are poorly known. To explore the utility of the stable hydrogen isotope approach, we examined variation in δD in stream food webs in a forested catchment where variation in δ¹³C has been described previously. 3. Although algal δD varied by taxa and, to a small degree, between sites, we found consistent and clear separation (by an average of 67‰) from terrestrial carbon sources. Environmental conditions known to affect algal δ¹³C, such as water velocity and stream productivity did not greatly influence algal δD, and there was no evidence of seasonal variation. In contrast, algal δ¹³C was strongly affected by environmental factors both within and across sites, was seasonally variable at all sites, and partially overlapped with terrestrial δ¹³C in all streams with catchment areas larger than 10 km². 4. While knowledge of isotopic exchange with water and trophic fractionation of δD for aquatic consumers is limited, consistent source separation in streams suggests that δD may provide a complementary food web tracer to δ¹³C in aquatic food webs. Lack of significant seasonal or spatial variation in δD is a distinct advantage over δ¹³C for applications in many aquatic ecosystems.     

Using stable hydrogen isotopes (δ2H) and ring recoveries to trace natal origins in a Eurasian passerine with a migratory divide

Despite recent advances in technology, it remains difficult to connect breeding and non‐breeding areas of populations of migratory organisms due to the challenges of year‐round tracking. Here, we used the Eurasian reed warbler Acrocephalus scirpaceus, a passerine with a pronounced migratory divide to demonstrate the promise of integrating several sources of information within the Bayesian modelling framework for the study of migratory connectivity. To this end, we combined data from stable hydrogen isotope ratios (δ²H) of feathers, ring recoveries, and the geographic delineation of sub‐populations on either side of the migratory divide. Feather δ²H measurements from local juvenile birds sampled across the breeding range tightly correlated with amount‐weighted mean annual precipitation δ²H values predicted for the natal sites. Predicted natal origins of birds intercepted en route in the Mediterranean region largely differed among the five stopover sites. Thanks to the different migratory pathways used by different breeding populations and the existence of a migratory divide, we were able to effectively narrow the assigned regions of origin. Our results show that spatial resolution of likelihood‐based assignments of geographic origins based on δ²H measurements may improve significantly when prior probabilities derived from population‐specific migratory directions are included. Integrating information from stable isotopes, ring recoveries, geolocators and other sources within the Bayesian modelling framework will provide an extremely useful toolbox for the study of animal movements in the future.     

Linking breeding and wintering grounds of neotropical migrant songbirds using stable hydrogen isotopic analysis of feathers

 Recent studies have shown that stable hydrogen isotope ratios (δD) in the tissues of animals often correlate with δD of local precipitation. Here we examined the relationship between δD in feathers and growing season precipitation for neotropical migrant songbirds breeding over a continent-wide isotopic gradient. δD values were determined on feathers of 140 individuals of 6 species of wild insectivorous forest songbirds (Setophaga ruticilla, Empidonax minimus, Vermivora peregrinus, Catharus ustulatus, Seiurus aurocapillus, Hylocichla mustelina) taken from 14 breeding locations across North America. The δD of feathers was strongly correlated with the δD of growing season precipitation at breeding sites across North America. As feather hydrogen is metabolically inert after growth, this relationship was then used to assess the breeding origins of wintering migrants. Deuterium values of feathers from 64 individuals representing 5 species of migrants (Helmitheros vermivorus, Wilsonia citrina, Hylocichla mustelina, Dumetella carolinensis, Seirus aurocapillus) at a wintering site in Guatemala were consistent with those predicted from the known breeding ranges of these species. This study demonstrates hydrogen isotopes may become a powerful tool for linking breeding and wintering grounds of neotropical migrant songbirds, as well as other migratory species moving between isotopically distinct regions. Received: 25 August 1995 / Accepted: 28 May 1996     

Metabolic routing of dietary nutrients in birds: effects of diet quality and macronutrient composition revealed using stable isotopes

During fall migration many songbirds switch from consuming primarily insects to consuming mostly fruit. Fruits with more carbohydrates and less protein may be sufficient to rebuild expended fat stores, but such fruits may be inadequate to replace catabolized protein. We manipulated the concentrations and isotopic signatures of macronutrients in diets fed to birds to study the effects of diet quality on metabolic routing of dietary nutrients. We estimated that approximately 45% and 75%, respectively, of the carbon in proteinaceous tissue of birds switched to high- or low-protein diets came from nonprotein dietary sources. In contrast, we estimated that approximately 100% and 20%-80%, respectively, of the nitrogen in proteinaceous tissues of birds switched to high- or low-protein diets was attributable to dietary protein. Thus, the routing and assimilation of dietary carbon and nitrogen differed depending on diet composition. As a result, delta (15)N of tissues collected from wild animals that consume high-quality diets may reliably indicate the dietary protein source, whereas delta (13)C of these same tissues is likely the product of metabolic routing of carbon from several macronutrients. These results have implications for how isotopic discrimination is best estimated and how we can study macronutrient routing in wild animals.