GENETICS: BIRDS
Strausberger B. M., Ashley M. V. (2001): Eggs yield nuclear DNA from egg-laying female cowbirds, their embryos and offspring. Conservation Genetics 2: 385-390.
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Here we report methods for extracting maternal DNA from avian eggshells or offspring DNA from eggshells and embryos. These methods offer alternative techniques for obtaining DNA from oviparous organisms. Using DNA extracted from eggshells, we obtain microsatellite genotypes of the brood parasitic brown-headed cowbird (Molothrus ater) female that laid the eggs and/or her hatched offspring. Using DNA extracted from embryos, we obtain microsatellite genotypes of offspring. We demonstrate that separate extractions performed on the embryo and shell from a single egg can provide DNA from the embryo and its mother, respectively. This single-egg approach for obtaining both maternal and embryonic DNA simplifies paternity analyses because alleles unique to the embryo can be considered paternal in origin. Finally we report two new microsatellite loci and primer sequences for brown-headed cowbirds.
Segelbacher G. (2002): Noninvasive genetic analysis in birds: testing reliability of feather samples. Molecular Ecology Notes 2: 367-369.
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Noninvasive samples are useful for molecular genetic analysis of free‐ranging animals. I tested whether moulted feathers collected in the field are a reliable source of DNA for genotyping microsatellite loci. I prescreened extracts for DNA quantity and, using only samples with higher amounts of DNA, obtained reliable genotyping results. Polymerase chain reaction (PCR) amplification success was higher from extracts of plucked feathers than moulted feathers. DNA quantity in larger feathers was higher than that in smaller feathers. This study clearly demonstrates that moulted feathers could be used for genetic studies in birds.
Idaghdour Y., Broderick D., Korrida A. N. D. A. (2003): Faeces as a source of DNA for molecular studies in a threatened population of great bustards. Conservation Genetics 4: 789-792.
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With recent advances in molecular biology, it is now possible to use the trace amounts of DNA in faeces to non-invasively sample endangered species for genetic studies. A highly vulnerable population of approximately 100 great bustards (Otis tarda) exists in Morocco necessitating the use of non-invasive protocols to study their genetic structure. Here we report a reliable silica-based method to extract DNA from great bustard faeces. We found that successful extraction and amplification correlated strongly with faeces freshness and composition. We could not extract amplifiable DNA from 30% of our samples as they were dry or contained insect material. However 100% of our fresh faecal samples containing no obvious insect material worked, allowing us to assess the levels of genetic variation among 25 individuals using a 542 bp control region sequence. We were able to extract DNA from four out of five other avian species, demonstrating that faeces represents a suitable source of DNA for population genetics studies in a broad range of species.
Handel C. M., Pajot L. M., Talbot S. L., Sage G. K. (2006): Use of buccal swabs for sampling DNA from nestling and adult birds. Wildlife Society Bulletin 34: 1094-1100.
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We evaluated the feasibility and efficiency of using swabs to collect buccal epithelial cells from small (2‐ to 13‐g) birds as a source of DNA for genetic studies. We used commercially available buccal swab kits to collect samples from 42 adult and 39 nestling (4‐ to 8‐day‐old) black‐capped chickadees (Poecile atricapillus) and from 6 4‐day‐old nestling boreal chickadees (P. hudsonica). We compared DNA from buccal epithelial samples to that from blood samples from the same individuals. We extracted sufficient quantities of DNA for analysis from all buccal samples, and samples remained viable even after being stored in original plastic sampling tubes at room temperature for up to 18 months. Yields were equivalent whether extracted using the proprietary quick‐extraction solution provided with buccal swab kits or using a salt‐extraction process with inexpensive reagents. Yields of DNA from buccal samples were consistently lower than those from blood samples, but quantities were sufficient for all analyses. Assignment of sex, based on DNA extracted from paired buccal and blood samples, was identical for all 87 birds. We found no difference in the genotypes obtained from buccal and blood samples for 12 individuals tested using 5 microsatellite loci and found perfect concordance in sequencing of an 823‐base‐pair segment within the control region of mitochondrial DNA for 7 individuals tested. Use of buccal swabs is highly recommended as a rapid, noninvasive technique for sampling avian genomic DNA, especially for extremely young altricial nestlings or small‐bodied adults, or for any birds for which blood sampling may be impossible or stressful.
Schmaltz G., Somers C. M., Sharma P., Quinn J. S. (2006): Non-destructive sampling of maternal DNA from the external shell of bird eggs. Conservation Genetics 7: 543-549.
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The use of non-destructive sampling methods to collect genetic material from wildlife allows researchers to minimize disturbance. Most avian studies employ capturing and handling of young and parents to draw blood for DNA analysis. In some cases adult female birds are difficult to catch, so maternal genotyping has required collection of contour feathers from nests, or destructive sampling of eggs. Many species do not leave contour feathers in the nest, and destructive sampling has been unreliable due to contamination with embryonic DNA. Alternative field sampling techniques for collection of maternal DNA from birds are therefore desirable. Here we demonstrate that avian maternal DNA can be isolated in a non-invasive and non-destructive way from the external surface of eggs. We used cotton swabs to collect maternal DNA from the external shells of herring gull (Larus argentatus) and Caspian tern (Sterna caspia) eggs. DNA was then amplified by the polymerase chain reaction (PCR) for microsatellite genotyping. We verified that the DNA samples were maternal by comparing microsatellite profiles to those obtained from adults and chicks from the same nests. In 100% of Caspian tern (n=16) and herring gull families (n=12), the egg swabs that amplified matched the maternal microsatellite genotype. In a screening of many nests of both species, we successfully amplified microsatellite markers from 101/115 (88%) egg swabs. Swabs from eggs with blood stains on the shell were more likely to amplify successfully than those from clean eggs. The advantages of this new method include increased parentage assignment/exclusion power, and increased availability of maternal DNA for genotyping of species that do not deposit contour feathers in nests.
Rudnick J. A., Katzner T. E., Bragin E. A., DeWoody J. A. (2007): Species identification of birds through genetic analysis of naturally shed feathers. Molecular Ecology Notes 7: 757-762.
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Genetic analysis of noninvasively collected bird feathers is of growing importance to avian ecology; however, most genetic studies that utilize feathers make no mention of the need to verify their species of origin. While plumage patterns and collection location often are indicative of species identity, broad‐scale feather collections may require definitive species identification prior to analysis. Genetic species identification has been applied to noninvasively collected samples from a wide range of taxa but, to date, these techniques have not been widely used on bird feathers. Here, we develop and test a polymerase chain reaction (PCR)‐based technique for identifying eastern imperial eagle (Aquila heliaca) samples among a vast number of noninvasively collected feathers. Species identification is accomplished by amplifying a fragment of the mitochondrial cytochrome c oxidase I gene, then digesting that fragment with a restriction enzyme. The resulting species‐specific restriction fragment length polymorphisms (RFLPs) are easily visualized by gel electrophoresis. We tested this PCR‐RFLP assay on over 300 individuals that had been genetically identified from noninvasively collected feathers and demonstrated that the assay is both reliable and robust for DNA of low quality and quantity. The genetic methods of species identification used to develop this assay can readily be applied to other bird assemblages, making them particularly relevant to a broad range of future avian research.
De Volo S. B., Reynolds R. T., Douglas M. R., Antolin M. F. (2008): An improved extraction method to increase DNA yield from molted feathers. The Condor 110: 762-766.
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To assess the value of molted feathers as a noninvasive source of DNA for genetic studies of Northern Goshawks (Accipiter gentilis), we isolated and quantified DNA from molted feathers and compared yields across five feather types. We also compared PCR success across the same five feather types using five microsatellite genetic markers of varying size. In addition, we compared DNA yields from a commonly used extraction method versus one we modified to increase DNA yield. Results indicated molted feathers provided on average 24 ng μl−1 of DNA, which is a relatively high DNA yield compared to other noninvasive tissue sources. Tail feathers yielded significantly more DNA than primary, secondary, and smaller feathers, yet all feather sizes produced equally high rates of PCR success. Although our modified extraction method increased the time required for processing feathers, it resulted in significantly higher yields of DNA as compared to the unmodified protocol.
Egloff C., Labrosse A., Hebert C., Crump D. (2009): A nondestructive method for obtaining maternal DNA from avian eggshells and its application to embryonic viability determination in herring gulls (Larus argentatus). Molecular Ecology Resources 9: 19-27.
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Many avian studies, aimed at collecting samples for genetic analysis, rely upon invasive procedures involving the capture and handling of parents and their offspring. Our goal was to develop a nondestructive method for sampling maternal DNA that would not require blood collection from the mother. Herein, we describe a method for isolating genomic DNA from eggshell powder, obtained by filing the outer shell of an avian egg. Comparison of microsatellite profiles, obtained from genomic DNA found within eggshell matrices and their corresponding parents, verified the presence of maternal DNA in the eggshell matrix in 100% of the herring gull nests assessed (n = 11). In addition, the microsatellite profiles of eggshell DNA were identical among eggs from the same clutch. The ability to rapidly obtain a DNA sample from an avian eggshell in a noninvasive manner could aid in a wide range of genetic sampling studies, and in this study, we provide one potential application of this finding: assessing the fertilization status of nonviable herring gull (Larus argentatus) eggs from the Laurentian Great Lakes. Detection of fertilization was successful as the microsatellite profiles of eggshell powder (maternal only) and the fertilized embryonic contents of those eggs did not match. Ideally, the application of such an approach will help to discriminate unfertilized eggs from embryos aborted early in development and provide insights into avian reproductive health.
Gebhardt K. J., Brightsmith D., Powell G., Waits L. P. (2009): Molted feathers from clay licks in Peru provide DNA for three large macaws (Ara ararauna, A. chloropterus, and A. macao). Journal of Field Ornithology 80: 183-192.
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Conservation genetic analyses of wildlife have increased greatly in the past 10 yr, yet genetic studies of parrots are rare because of difficulties associated with capturing them and obtaining samples. Recent studies have demonstrated that molted feathers can provide a useful source of DNA, but success rates have varied considerably among studies. Our objective was to determine if molted macaw feathers from Blue‐and‐yellow Macaws (Ara ararauna ), Scarlet Macaws (A. macao), and Red‐and‐green Macaws (A. chloropterus) collected from rainforest geophagy sites called clay licks could provide a good source of DNA for population genetic studies. Specific objectives were to determine (1) how nuclear DNA microsatellite amplification success and genotyping error rates for plucked macaw feathers compared to those for molted feathers collected from clay licks in the Amazon rainforest, and (2) if feather size, feather condition, species, or extraction method affected microsatellite amplification success or genotyping error rates from molted feathers. Amplification success and error rates were calculated using duplicate analyses of four microsatellite loci. We found that plucked feathers were an excellent source of DNA, with significantly higher success rates (P < 0.0001) and lower error rates (P = 0.0002) than for molted feathers. However, relatively high success rates (75.6%) were obtained for molted feathers, with a genotyping error rate of 11.7%. For molted feathers, we had higher success rates and lower error rates for large feathers than small feathers and for feathers in good condition than feathers that were moldy and broken when collected. We also found that longer incubation times and lower elution volumes yielded the highest quality DNA when extracting with the Qiagen DNeasy tissue kit. Our study demonstrates that molted feathers can be a valuable source of genetic material even in the challenging conditions of tropical rainforests, and our results provide valuable information for maximizing DNA amplification success rates when working with shed feathers of parrots.
Guerrini M., Barbanera F. (2009): Noninvasive genotyping of the red-legged partridge (Alectoris rufa, Phasianidae): semi-nested PCR of mitochondrial DNA from feces. Biochemical Genetics 47: 873.
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DNA-based studies using avian feces are scarce and deal only with large-sized species. The red-legged partridge, Alectoris rufa, is a medium-sized member of the order Galliformes. Our goal was to set up a fast, noninvasive procedure for mitochondrial DNA (mtDNA) genotyping of A. rufa fecal samples. We focused on the protected population from Elba Island (Tuscan Archipelago National Park, Italy). Dry A. rufa fecal samples (n = 30) were collected in winter. Both the cytochrome b gene (1,092 bp) and the control region (ca. 1,155 bp) were amplified by means of semi-nested PCRs. Twenty-five samples were successfully sequenced for both genes: 8 showed A. rufa mtDNA lineage and 17 chukar partridge (A. chukar), an exotic species. Mixed maternal ancestry suggests A. rufa × A. chukar hybridization. Our protocol allows noninvasive mtDNA genotyping of any Alectoris species and appears suitable to investigate protected populations as well as those existing either at very low density or inhabiting poorly accessible regions.
Marrero P., Fregel R., Cabrera V. M., Nogales M. (2009): Extraction of high-quality host DNA from feces and regurgitated seeds: a useful tool for vertebrate ecological studies. Biological Research 42: 147-151.
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DNA extraction methods for genotyping non-invasive samples have led to great advances in molecular research for ecological studies, and have been particularly useful for analyzing threatened species. However, scarce amounts of fragmented DNA and the presence of Taq polymerase inhibitors in non-invasive samples are potential problems for subsequent PCR amplifications. In this study we describe a novel technique for extracting DNA from alimentary tract cells found on external surfaces of feces and regurgitated seeds. The presence of contaminants and inhibitors is minimized and samples are preserved intact for use in other ecological research (e.g. trophic studies). The amplification efficiency and purity of the extracted DNA from feces were significantly higher than in commonly used extraction procedures. Moreover, DNA of two bird species was identified from seeds expelled by regurgitation. Therefore, this method may be suitable for future ecological studies of birds, and other vertebrate groups.
Miño C. I., Del Lama S. N. (2009): Molted feathers as a source of DNA for genetic studies in waterbird populations. Waterbirds 32: 322-329.
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The determination of genetic parameters for most waterbird populations presents considerable challenges because these species frequently breed in inaccessible locations which preclude capture of nesting pairs. The application of non-invasive sampling techniques, such as the collection of molted feathers in the field, could represent an appealing strategy to overcome the above cited problem. Here, the feasibility of sampling molted feathers collected in Brazilian populations of the Roseate Spoonbill (Platalea ajaja; N = 94) and the jabiru Stork (Jabiru mycteria; N = 10) for genetic studies was evaluated. Each feather yielded high quality DNA with a concentration 1.25 times higher than that obtained from feathers from similar sized birds from other groups. Amplification of a 390– 396 bp fragment for molecular sexing and 11 microsatellite loci was successful. The average percentage of positive PCRs (i.e. those that yielded products) among all Roseate Spoonbill loci and samples was 95.5%. The sampling approach appears suitable to gather genetic information in natural populations of these waterbirds and can be applied to other similar species.
Trimbos K. B., Broekman J., Kentie R., Musters C. J., de Snoo G. R. (2009): Using eggshell membranes as a DNA source for population genetic research. Journal of Ornithology 150: 915-920.
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In the context of population genetic research, a faster and less invasive method of DNA sampling would allow large-scale assessments of genetic diversity and genetic differentiation with the help of volunteer observers. The aim of this study was to investigate the usefulness of eggshell membranes as a DNA source for population genetic research, by addressing eggshell membrane DNA quality, degeneration and cross-contamination. To this end, a comparison was made with blood-derived DNA samples. We have demonstrated 100% successful DNA extraction from post-hatched Black-tailed Godwit (Limosa limosa) eggshell membranes as well as from blood samples. Using 11 microsatellite loci, DNA amplification success was 99.1% for eggshell membranes and 97.7% for blood samples. Genetic information within eggshell membrane DNA in comparison to blood DNA was not affected (FST = −0.01735, P = 0.999) by degeneration or possible cross-contamination. Furthermore, neither degeneration nor cross-contamination was apparent in total genotypic comparison of eggshell membrane DNA and blood sample DNA. Our research clearly illustrates that eggshell membranes can be used for population genetic research.
Maurer G., Beck N., Double M. C. (2010): A ‘feather‐trap’ for collecting DNA samples from birds. Molecular Ecology Resources 10: 129-134.
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Genetic analyses of birds are usually based on DNA extracted from a blood sample. For some species, however, obtaining blood samples is difficult because they are sensitive to handling, pose a conservation or animal welfare concern, or evade capture. In such cases, feathers obtained from live birds in the wild can provide an alternative source of DNA. Here, we provide the first description and evaluation of a ‘feather‐trap’, consisting of small strips of double‐sided adhesive tape placed close to a nest with chicks, as a simple, inexpensive and minimally invasive method to collect feathers. The feather‐trap was tested in tropical conditions on the Australian pheasant coucal (Centropus phasianinus). None of the 12 pairs of coucals on which the feather‐trap was used abandoned the nest, and feeding rates did not differ from those of birds not exposed to a feather‐trap. On average, 4.2 feathers were collected per trap over 2–5 days and, despite exposure to monsoonal rain, DNA was extracted from 71.4% of samples, albeit at low concentrations. The amount of genomic DNA extracted from each feather was sufficient to reliably genotype individuals at up to five microsatellite loci for parentage analysis. We show that a feather‐trap can provide a reliable alternative for obtaining DNA in species where taking blood is difficult. It may also prove useful for collecting feather samples for other purposes, e.g. stable‐isotope analysis.
Martín-Gálvez D., Peralta-Sánchez J. M., Dawson D. A., Martín-Platero A. M., Martínez-Bueno M., Burke T., Soler J. J. (2011): DNA sampling from eggshell swabbing is widely applicable in wild bird populations as demonstrated in 23 species. Molecular Ecology Resources 11: 481-493.
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There is increasing interest in noninvasive DNA sampling techniques. In birds, there are several methods proposed for sampling DNA, and of these, the use of eggshell swabbing is potentially applicable to a wide range of species. We estimated the effectiveness of this method in the wild by sampling the eggs of 23 bird species. Sampling of eggs was performed twice per nest, soon after the clutch was laid and again at the end of egg incubation. We genotyped DNA samples using a set of five conserved microsatellite markers, which included a Z‐linked locus and a sex‐typing marker. We successfully collected avian DNA from the eggs of all species tested and from 88.48% of the samples. In most of the cases, the DNA concentration was low (ca. 10 ng/μL). The number of microsatellite loci amplified per sample (0–5) was used as a measure of the genotyping success of the sample. On average, we genotyped 3.01 ± 0.12 loci per sample (mean ± SE), and time of sampling did not seem to have an effect; however, genotyping success differed among species and was greater in those species that used feather material for lining their nest cups. We also checked for the occurrence of possible genotyping errors derived from using samples with very low DNA quantities (i.e. allelic dropout or false alleles) and for DNA contamination from individuals other than the mother, which appeared at a moderate rate (in 44% of the PCR replicates and in 17.36% of samples, respectively). Additionally, we investigated whether the DNA on eggshells corresponded to maternal DNA by comparing the genotypes obtained from the eggshells to those obtained from blood samples of all the nestlings for six nests of magpies. In five of the six magpie nests, we found evidence that the swab genotypes were a mixture of genotypes from both parents and this finding was independent of the time of incubation. Thus, our results broadly confirm that the swabbing of eggshells can be used as a noninvasive method for obtaining DNA and is applicable across a wide range of bird species. Nonetheless, genotyping errors should be properly estimated for each species by using a suite of highly polymorphic loci. These errors may be resolved by sampling only recently laid eggs (to avoid non‐maternal DNA contamination) or by performing several PCR replicates per sample (to avoid allelic dropout and false alleles) and/or by increasing the amount of DNA used in the PCR through increasing the volume of the PCR or increasing the concentration of template DNA.
Kjelland M. E., Kraemer D. (2012): Feathers and post-hatch eggshells: sources of fibroblast cells for conserving genetic diversity. Avian Biology Research 5: 123-130.
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Scientific literature addresses the use of feathers in sex determination, as a source of DNA, and for providing ecotoxicological information; however, there is much less research on the use of feathers as a non-invasive, or minimally invasive, means to conserve avian genetic diversity. This study investigated the use of semi-mature to mature feathers and post-hatch eggshells, as well as embryos, as sources of fibroblast-like cells for cytological analysis and somatic cell gene banking. Contour and flight feathers were plucked from living Rhode Island red and white leghorn varieties of the domestic chicken (Gallus domesticus), India blue peafowl (white mutation) (Pavo cristatus), domestic turkey (Meleagris gallopavo), domestic duck (Anas platyrhyncha), emu (Dromaius novaehollandiae), and ostrich (Struthio camelus). Fibroblast cell growth was observed in the feather pulp and post-hatch eggshell samples after 24-48 hours. Erythrocytes, macrophages, and epithelial cells were also observed, based on morphology. Semi-mature to mature feathers containing feather pulp and post-hatch eggshells can be adequate sources of fibroblast cells for somatic cell acquisition and cell culture, possibly providing a source of cells for use in chimera formation or cloning of threatened and endangered birds. Further research should focus on applying this technique to other avian species and address cryopreservation and viability of cells derived in this manner.
Wellbrock A. H., Bauch C., Rozman J., Witte K. (2012): Buccal swabs as a reliable source of DNA for sexing young and adult Common Swifts (Apus apus). Journal of Ornithology 153: 991-994.
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We investigated the reliability of buccal swabs for molecular sex determination in very young nestlings (0–14 days old) and adults of the Common Swift (Apus apus) compared to the results from blood samples of the same individuals. Sex determination based on buccal swabs matched the result of sex determination based on blood samples in 46 out of 47 nestlings (98 %) and in all 10 adults (100 %). Therefore, we consider that buccal swab sampling is a reliable noninvasive method to obtain DNA for sex determination in swifts. We recommend buccal swabbing as an alternative to blood sampling in future genetic studies in birds.
Vili N., Nemesházi E., Kovács S., Horváth M., Kalmár L., Szabó K. (2013): Factors affecting DNA quality in feathers used for non-invasive sampling. Journal of Ornithology 154: 587-595.
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The development of genetic methods broadens the scope of non-invasive sample types. Large shed feathers are good material for genetic analyses, since they are easy to collect and a single feather can provide a sufficient amount of DNA for PCR-based methods. Previous studies have demonstrated that feather quality and type affect the DNA quality extracted from the feather tips. Besides the tip, the superior umbilicus part of the shaft is also proposed as an appropriate source of DNA. In our study, we examined whether some feather parameters (physical condition, type and size) and storage time affect amplification success of DNA extracted from the superior umbilicus of shed Eastern Imperial Eagle (Aquila heliaca) feathers. We also tested the effects of sunlight, temperature and humidity on DNA extracted from Domestic Goose (Anser anser domesticus) feathers with amplification of fragments of various sizes, modelling the environmental conditions of the moulting season. While good quality feathers usually provided sufficient DNA, the usability of the DNA extracted from moderate quality feathers were affected by feather type. DNA quality was influenced in order of importance by humidity, direct sunlight and heat. Our findings support the usability of DNA samples derived from the superior umbilicus of shed feathers, and help to schedule field work by careful consideration of our results about feather quality and environmental factors.
Adam I., Scharff C., Honarmand M. (2014): Who is who? Non-invasive methods to individually sex and mark altricial chicks. JoVE 87: e51429.
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Many experiments require early determination of offspring’s sex as well as early marking of newborns for individual recognition. According to animal welfare guidelines, non-invasive techniques should be preferred whenever applicable. In our group, we work on different species of song birds in the lab and in the field, and we successfully apply non-invasive methods to sex and individually mark chicks. This paper presents a comprehensive non-invasive tool-box. Sexing birds prior to the expression of secondary sexual traits requires the collection of DNA-bearing material for PCR. We established a quick and easy method to sex birds of any age (post hatching) by extracting DNA from buccal swabs. Results can be obtained within 3 hours. For individual marking chick’s down feathers are trimmed in specific patterns allowing fast identification within the hatching order. This set of methods is easily applicable in a standard equipped lab and especially suitable for working in the field as no special equipment is required for sampling and storage. Handling of chicks is minimized and marking and sexing techniques are non-invasive thereby supporting the RRR-principle of animal welfare guidelines.
Rösner S., Brandl R., Segelbacher G., Lorenc T., Müller J. (2014): Noninvasive genetic sampling allows estimation of capercaillie numbers and population structure in the Bohemian Forest. European Journal of Wildlife Research 60: 789-801.
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Wildlife conservation and management of endangered species requires reliable information on the size and structure of populations. One of the flagship species in European wildlife conservation is the forest-dwelling capercaillie (Tetrao urogallus), where several populations are endangered. In the Bohemian Forest, e.g., the population severely declined 30 years ago with only 100 birds remaining in 1985. Subsequently, breeding and release programs were conducted to supplement the local population. The current distribution and population size, however, remained unknown. With recent habitat changes and increasing recreational activities, a reliable population estimate to inform conservation plans was needed. A team of scientists and volunteers collected fresh capercaillie droppings covering an area of about 120,000 ha. We genotyped ten microsatellite loci to estimate the current population size and to determine the population’s spatial and genetic structure. Population size and density estimators revealed a population size of approximately 500 individuals, which is thus one of the two largest relict populations in the low mountain ranges of temperate Europe. The population clustering revealed gene flow across the entire study area. Several genotypes were documented with multiple recaptures at spatial distances between 10 and 30 km additionally corroborating gene flow across the entire landscape of the study area. Males were more closely related than females on small spatial scales up to 3 km, indicating lower dispersal rates in males. We conclude that the population currently appears to have a viable size and shows unrestricted gene flow across state borders and management units of the entire Bohemian Forest. However, long-term viability of this population requires a transboundary strategy to sustainably protect and monitor this isolated capercaillie population in Central Europe.
Dai Y., Lin Q., Fang W., Zhou X., Chen X. (2015): Noninvasive and nondestructive sampling for avian microsatellite genotyping: a case study on the vulnerable Chinese egret (Egretta eulophotes). Avian Research 6: 24.
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Noninvasive and nondestructive DNA sampling techniques are becoming more important in genetic studies because they can provide genetic material from wild animals with less or even without disturbance, which is particularly useful for the study of endangered species, i.e., birds. However, nondestructively and noninvasively sampled DNA may, in some cases, be inadequate in the amount and quality of the material collected, which can lead to low amplification success rates and high genotyping errors. In this study, noninvasive (eggshell swab, shed feather and feces), nondestructive (plucked feather and buccal swab) and invasive (blood) DNA samples were collected from the vulnerable Chinese egret (Egretta eulophotes). DNA concentrations, PCR amplification success and microsatellite genotyping errors of different sample types were evaluated and compared to determine whether noninvasive and nondestructive samples performed as well as invasive samples in our experimental procedures. A total of 159 samples were collected in the field. Among the different sample types, the highest DNA concentrations (154.0–385.5 ng/μL) were obtained from blood. Those extracted from fecal samples were the lowest, ranging from 1.25 to 27.5 ng/μL. Almost all of the DNA samples, i.e., 95.59 %, were successfully amplified for mtDNA (n = 152) and 92.76 % of mtDNA samples were successfully genotyped for at least five of the nine microsatellite loci tested (n = 141). Blood samples and buccal swabs produced reliable genotypes with no genotyping errors, but in feces, allelic dropouts and false alleles occurred in all nine loci, with error rates ranging from 6.67 to 38.10 % for the dropouts and from 6.06 to 15.15 % for the false alleles. These results indicate that both nondestructive and noninvasive samplings are suitable for avian microsatellite genotyping, save for fecal DNA. However, we should remain cautious of the appearance of genotyping errors, especially when using noninvasive material.
Olah G., Heinsohn R. G., Brightsmith D. J., Espinoza J. R., Peakall R. (2016): Validation of non-invasive genetic tagging in two large macaw species (Ara macao and A. chloropterus) of the Peruvian Amazon. Conservation Genetics Resources 8: 499-509.
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Genetic tagging is the unique identification of individuals by their DNA profile. This technique is well established in mammals, but it has not yet been widely adopted for birds. Extraction methods for minute amounts of DNA even enable the use of genetic tagging from non-invasive samples, like hair, scat, or feather. In this study, we evaluate the potential for non-invasive genetic tagging by using molted feathers of two sympatric macaw species in the Peruvian Amazon. Correct species identification is critical when relying on feathers for genetic analysis, so we describe multilocus methods for species identification. We evaluate the quality of naturally shed macaw feathers in tropical environmental conditions and present new primers for molecular sexing on the feather samples. We successfully validated 11 microsatellite markers for use in genetic tagging studies on large macaws and confirmed that DNA from blood and feather samples yields equivalent population genetic patterns. The techniques described here can be implemented for other birds with higher conservation concern.
Begovic L., Mihic I., Pospihalj T., Mikuska T., Mlinaric S., Mikuska A. (2017): Evaluation of methods for molecular sex-typing of three heron species from different DNA sources. Turkish Journal of Zoology 41: 593-598.
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Genetic markers are a useful tool for bird population monitoring, especially when combined with ringing data, and particularly so in vulnerable species. This study compared the effectiveness of two DNA extraction methods: a standard protocol and a commercially available kit. The molecular sex-typing was performed using PCR-based methods with a 2550F/2718R set of primers in three species of herons: Great Egret, Purple Heron, and Grey Heron. Genomic DNA was isolated from feathers, eggshells, and eggshell swabs from 26 individuals. Overall, better DNA yields and purity were obtained by using the standard protocol isolation method. The highest DNA yield was obtained from the pin feathers compared to the contour feathers and eggshells, both of which had lower yields. Eggshell swabs indicated possible contamination with parental/sibling DNA. Our evaluation demonstrates that the optimization of laboratory procedures is beneficial, particularly when different types of noninvasive tissue samples are available.
Maia T. A., Vilaça S. T., Silva L. R. D., Santos F. R., Dantas G. P. D. M. (2017): DNA sampling from eggshells and microsatellite genotyping in rare tropical birds: case study on Brazilian Merganser. Genetics and Molecular Biology 40: 808-812.
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This study shows that sampling maternal DNA from hatched and abandoned eggshells is a viable noninvasive strategy for studying the genetics of rare or endangered tropical birds, as exemplified here by the Brazilian Merganser (Mergus octosetaceus). Eighteen microsatellites were isolated from enriched libraries and nine heterologous loci from related species were tested. Seven loci were amplified successfully, with five of them being polymorphic. These loci exhibited amplicons ranging from 110 to 254 bp for 132 samples, with 60 from eggshells and 72 from blood or muscle samples. The number of alleles for M. octosetaceus ranged from one to six (mean = 3.71), which is low compared to M. merganser (1-15 alleles), a ‘least concern’ species. Genetic diversity did not differ significantly between noninvasive and invasive samples (Z(u) = 0.31, p = 0.37). Thus, noninvasive sampling, as demonstrated here with eggshells, provides an efficient means to assess genetic diversity in tropical birds without the need to capture and handle them.
Olah G., Heinsohn R. G., Brightsmith D. J., Peakall R. (2017): The application of non-invasive genetic tagging reveals new insights into the clay lick use by macaws in the Peruvian Amazon. Conservation Genetics 18: 1037-1046.
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Genetic tagging, the unique identification of individuals by their DNA profile, has proven to be an effective method for research on several animal species. In this study we apply non-invasive genetic tagging from feather samples to reveal the genetic structure and estimate local population size of red-and-green macaws (Ara chloropterus) without the need to capture these animals. The study was centered in the Tambopata region of the Peruvian Amazon. Here macaws frequently visit clay licks and their naturally molted feathers provide a unique source of non-invasively sampled DNA. We analyzed 249 feathers using nine microsatellite loci and identified 221 unique genotypes. The remainder revealed 21 individuals which were ‘recaptured’ one or more times. Using a capture-mark-recapture model the average number of different individuals visiting clay licks within one breeding season was estimated to fall between 84 and 316 individuals per clay lick. Analysis of population genetic structure revealed only small genetic differences among regions and clay licks, suggesting a single red-and-green macaw genetic population. Our study confirms the utility of non-invasive genetic tagging in harsh tropical environment to obtain crucial population parameters about an abundant parrot species that is very difficult to capture in the wild.
Ramón‐Laca A., White D. J., Weir J. T., Robertson H. A. (2018): Extraction of DNA from captive‐sourced feces and molted feathers provides a novel method for conservation management of New Zealand kiwi (Apteryx spp.). Ecology and Evolution 8: 3119-3130.
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Although some taxa are increasing in number due to active management and predator control, the overall number of kiwi (Apteryx spp.) is declining. Kiwi are cryptic and rare, meaning current monitoring tools, such as call counts, radio telemetry, and surveys using detection dogs are labor‐intensive, yield small datasets, and require substantial resources or provide inaccurate estimates of population sizes. A noninvasive genetic approach could help the conservation effort. We optimized a panel of 23 genetic markers (22 autosomal microsatellite loci and an allosomal marker) to discriminate between all species of kiwi and major lineages within species, while simultaneously determining sex. Markers successfully amplified from both fecal and shed feather DNA samples collected in captivity. We found that DNA extraction was more efficient from shed feathers, but DNA quality was greater with feces, although this was sampling dependent. Our microsatellite panel was able to distinguish between contemporary kiwi populations and lineages and provided PI values in the range of 4.3 × 10−5 to 2.0 × 10−19, which in some cases were sufficient for individualization and mark–recapture studies. As such, we have tested a wide‐reaching, noninvasive molecular approach that will improve conservation management by providing better parameter estimates associated with population ecology and demographics such as abundance, growth rates, and genetic diversity.
Vallant S., Niederstätter H., Berger B., Lentner R., Parson W. (2018): Increased DNA typing success for feces and feathers of capercaillie (Tetrao urogallus) and black grouse (Tetrao tetrix). Ecology and Evolution 8: 3941-3951.
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Noninvasive sampling, for example, of droppings or feathers, is a promising approach for molecular genetic studies on endangered and elusive animal species. Yet, such specimens are known for containing only minute amounts of DNA , resulting in lower typing success rates relative to analyses on fresh tissues such as muscle or blood. Furthermore, artefactual signals as well as contamination are more likely to occur when DNA is limited. To increase the reliability of DNA typing from noninvasive samples, optimized DNA extraction and polymerase chain reaction protocols were developed, taking advantage of developments in the forensic field aiming at successful molecular genetic analysis of DNA templates being low in quality and quantity. In the framework of an extensive monitoring project on population dynamics of capercaillie and black grouse in the Tyrolean Alps, feces samples and molted feathers from both species were collected. On a subset comprising about 200 specimens of either species, eight polymorphic short tandem repeat (STR) markers were analyzed to test these improved protocols. Besides optimizing DNA yields, both lowered sample consumption and reduced hands‐on time were achieved, and the rates of informative profiles amounted to 90.7% for capercaillie and 92.4% for black grouse. Similarly, high success rates had not been achieved in earlier studies and demonstrate the benefit of the improved methodology, which should be easily adaptable for use on animal species other than those studied here. The STR genotypes were not only powerful enough to discriminate among unrelated birds but also appeared fit for telling apart closely related animals, as indicated by Pi and Pisib values. The software package allelematch aided analysis of genotypes featuring possible dropout and drop‐in effects. Finally, a comparison between molecular genetic and morphology‐based species‐of‐origin determination revealed a high degree of concordance.
Vilstrup J. T., Mullins T. D., Miller M. P., McDearman W., Walters J. R., Haig S. M. (2018): A simplified field protocol for genetic sampling of birds using buccal swabs. The Wilson Journal of Ornithology 130: 326-334.
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DNA sampling is an essential prerequisite for conducting population genetic studies. For many years, blood sampling has been the preferred method for obtaining DNA in birds because of their nucleated red blood cells. Nonetheless, use of buccal swabs has been gaining favor because they are less invasive yet still yield adequate amounts of DNA for amplifying mitochondrial and nuclear markers; however, buccal swab protocols often include steps (e.g., extended air-drying and storage under frozen conditions) not easily adapted to field settings. Furthermore, commercial extraction kits and swabs for buccal sampling can be expensive for large population studies. We therefore developed an efficient, cost-effective, and field-friendly protocol for sampling wild birds after comparing DNA yield among 3 inexpensive buccal swab types (2 with foam tips and 1 with a cotton tip). Extraction and amplification success was high (100% and 97.2% respectively) using inexpensive generic swabs. We found foam-tipped swabs provided higher DNA yields than cotton-tipped swabs. We further determined that omitting a drying step and storing swabs in Longmire buffer increased efficiency in the field while still yielding sufficient amounts of DNA for detailed population genetic studies using mitochondrial and nuclear markers. This new field protocol allows time- and cost-effective DNA sampling of juveniles or small-bodied birds for which drawing blood may cause excessive stress to birds and technicians alike.
Monge O., Dumas D., Baus I. (2020): Environmental DNA from avian residual saliva in fruits and its potential uses in population genetics. Conservation Genetics Resources 12: 131–139.
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Buccal cells are a valid source of vertebrate DNA for genetic analysis, typically collected with mouth swabs on captured individuals. Yet, advances in DNA storage and processing now permit recovering genomic material from traces of saliva in food remains. For example, the feeding habits of canopy-dwelling frugivorous birds, e.g. large macaws (Psittaciformes), could present an ideal opportunity to obtain environmental DNA (eDNA) from partially consumed fruits. We tested this by collecting tropical almond (Terminalia catappa) fruits, eaten and discarded by scarlet macaws (Ara macao), and processed them using three different collection/storage methods. We successfully isolated DNA molecules from macaw residual saliva in fruits. This genetic material allowed the amplification of 7 microsatellite markers and of the CHD region of the avian sex chromosomes using two different primer pairs. Macaw eDNA concentration (mean 12 ng/µl) was similar to the reported in other avian studies using buccal swabs but overall microsatellite (60% success) and CHD-gen (20% success) amplification was low. The best results were obtained for samples preserved in ethanol. We conclude that saliva eDNA in partially consumed food items is an underused source of non-invasive bird DNA for genetic analyses. Based on our results, we recommend that a combination of specialized collection swabs or ethanol stored swabs along with commercial DNA extraction kits be used. Protocols should be modified accordingly to reach a consistent level of individual identification and gender determination that closely matches traditional sampling.
Peters C., Nelson H., Rusk B., Muir A. (2020): A novel method to optimise the utility of underused moulted plumulaceous feather samples for genetic analysis in bird conservation. Conservation Genetics Resources 12: 457–467.
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Non-invasive sampling methods are increasingly being used in conservation research as they reduce or eliminate the stress and disturbance resulting from invasive sampling of blood or tissue. Here we present a protocol optimised for obtaining usable genetic material from moulted plumulaceous feather samples. The combination of simple alterations to a ‘user-developed’ method, comprised of increased incubation time and modification of temperature and volume of DNA elution buffer, are outlined to increase DNA yield and significantly increase DNA concentration (W = 81, p < 0.01, Cohens’s d = 0.89). We also demonstrate that the use of a primerless polymerase chain reaction (PCR) technique increases DNA quality and amplification success when used prior to PCR reactions targeting avian mitochondrial DNA (mtDNA). A small amplicon strategy proved effective for mtDNA amplification using PCR, targeting three overlapping 314–359 bp regions of the cytochrome oxidase I barcoding region which, when combined, aligned with target-species reference sequences. We provide evidence that samples collected non-invasively in the field and kept in non-optimal conditions for DNA extraction can be used effectively to sequence a 650 bp region of mtDNA for genetic analysis.
Taylor D. R., Kluever B. M., Humphrey J. S., Hennessy I. M., Sutton A., Bruce W. E., Piaggio A. J. (2022): Amplification of Black Vulture (Coragyps atratus) DNA from regurgitated food pellets. The Wilson Journal of Ornithology 134: 546-551.
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Studies that rely on noninvasive collection of DNA for birds often use feces or feathers. Some birds, such as vultures, regurgitate undigested matter in the form of pellets that are commonly found under roost sites. Our research demonstrates that regurgitated pellets are a viable, noninvasive source of DNA for molecular ecology studies of vultures. Our objectives were to amplify 5 microsatellite loci designed for distinguishing Turkey Vultures (Cathartes aura) and Black Vultures (Coragyps atratus) in a single, multiplexed PCR, and to determine how long the target nuclear DNA persists after a vulture pellet is regurgitated and exposed to the environment. We collected pellets from captive Black Vultures and placed them in an outdoor aviary for a maximum estimated total of 12, 24, 36, or 48 h. We swabbed pellet surfaces for extraction and amplified vulture DNA using the panel of markers. All amplified alleles fell within predicted ranges of Black Vultures for all 5 loci, supporting the use of this microsatellite panel for vulture species identification. Overall amplification success for samples collected 0–12 h after regurgitation was 82.3%. Pellets collected 12–24 h, 24–36 h, and 36–48 h after regurgitation had only 18%, 10.2%, and 4.5% amplification success, respectively, which may have been due to a rain event. Our approach will be useful for noninvasive genetic sampling targeting nuclear DNA. These results should encourage noninvasive genetic sampling studies of other species that regurgitate pellets, such as raptors, water birds, or shorebirds.
Schweizer T. M., DeSaix M. G. (2023): Cost-effective library preparation for whole genome sequencing with feather DNA. Conservation Genetics Resources 15: 21-28.
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Samples from avian species of high conservation concern are often low in total genomic DNA (gDNA). Feathers are collected more frequently than blood, yet they contain low total gDNA which can present challenges in using them for genomics. Whole Genome Sequencing (WGS) can provide many insights that can be used to aid in species conservation, but current methods for working with feathers are cost prohibitive for population level genomic analyses. Thus, there is a need for a cost-effective method of preparing WGS libraries from feathers. To bridge the gap between sampling techniques commonly used in avian conservation genetics that yield low total gDNA and the powerful tool of WGS, we developed a method that successfully produces WGS libraries from feather gDNA with as low input as 0.48 ng of DNA and an average final library size of 300–500 base pairs. Sequencing results suggest high sequencing quality using our protocol with feather DNA. We conclude that our method will facilitate high-throughput WGS on low total gDNA samples, like feathers, thus expanding the power of genomic tools in critical avian conservation research.