Sex identification in embryos and adults of Darwin's finches.
ABSTRACT: Darwin's finches are an iconic example of adaptive radiation and evolution under natural selection. Comparative genetic studies using embryos of Darwin's finches have shed light on the possible evolutionary processes underlying the speciation of this clade. Molecular identification of the sex of embryonic samples is important for such studies, where this information often cannot be inferred otherwise. We tested a fast and simple chicken embryo protocol to extract DNA from Darwin's finch embryos. In addition, we applied minor modifications to two of the previously reported PCR primer sets for CHD1, a gene used for sexing adult passerine birds. The sex of all 29 tested embryos of six species of Darwin's finches was determined successfully by PCR, using both primer sets. Next to embryos, hatchlings and fledglings are also impossible to distinguish visually. This extends to juveniles of sexually dimorphic species which are yet to moult in adult-like plumage and beak colouration. Furthermore, four species of Darwin's finches are monomorphic, males and females looking alike. Therefore, sex assessment in the field can be a source of error, especially with respect to juveniles and mature monomorphic birds outside of the mating season. We caught 567 juveniles and adults belonging to six species of Darwin's finches and only 44% had unambiguous sex-specific morphology. We sexed 363 birds by PCR: individuals sexed based on marginal sex specific morphological traits; and birds which were impossible to classify in the field. PCR revealed that for birds with marginal sex specific traits, sexing in the field produced a 13% error rate. This demonstrates that PCR based sexing can improve field studies on Darwin's finches, especially when individuals with unclear sex-related morphology are involved. The protocols used here provide an easy and reliable way to sex Darwin's finches throughout ontogeny, from embryos to adults.
Project description:Accurate sexing of birds is vital for behavioral studies but can be a real problem in the field, especially for monomorphic species. Our goal here was to characterize the morphology of male and female monomorphic pale-winged starlings (Onychognathus nabouroup), a South African sturnid whose plumage is sexually monomorphic. Morphological measurements of genetically sexed animals indicated that males were statistically larger than females for five measurements: Mass, tail length, tarsus length and wing length. By using a Discriminant Function Analysis based on the measurements taken by one ringer, we were able to predict correctly the sex of 81.10% of the birds of data collected in the field and 77.9% of museum skins independently of year of capture and ringer. The model developed here should be useful for further field studies of this species.
Project description:Background:The sex of an individual organism plays such an important role in its life cycle that researchers must know a bird's sex to interpret key aspects of its biology. The sexes of dimorphic species can be easily distinguished, but sexing monomorphic bird species often requires expensive and time-consuming molecular methods. The Little Stint (Calidris minuta) is a numerous species, monomorphic in plumage but showing a small degree of reversed sexual size dimorphism. Females are larger than males but the ranges of their measurements overlap, making Little Stints difficult to sex in the field. Our aim was to develop reliable sexing criteria for Little Stints in different stages of primary moult during their stay on the non-breeding grounds in South Africa using DNA-sexed individuals and discriminant function analysis. Methods:We caught 348 adult Little Stints in 2008-2016 on their non-breeding grounds at Barberspan Bird Sanctuary. To molecularly identify the birds' sex we used P2/P8 primers and DNA isolated from blood samples collected in the field. We used Storer's dimorphism index to assess the degree of sexual size dimorphism. Then we divided our sample into two groups: before or during and after primary moult. For each group we developed two functions: one using wing length only and the other a combination of morphometric features including wing, tarsus and total head length. Then we used a stepwise procedure to check which combination of measurements best discriminated sexes. To validate our result we used a jack-knife cross-validation procedure and Cohen-kappa statistics. Results:All the morphometric features we measured were bigger in DNA-sexed females than in males. Birds with fresh primaries had on average 2.3 mm longer wings than those with worn primaries. A discriminant function using wing length (D1) correctly sexed 78.8% of individuals before moult, and a stepwise analysis showed that a combination of wing length and tarsus (D2) correctly identified the sex of 82.7% of these birds. For birds with freshly moulted primaries a function using wing length (D3) correctly classified 83.4% of the individuals, and a stepwise analysis revealed that wing and total head length (D4) classified 84.7%. Discussion:Sexual size differences in Little Stints might be linked to their phylogenetics and breeding biology. Females are bigger, which increases their fecundity; males are smaller, which increases their manoeuverability during display flights and hence their mating success. Little Stints show an extreme lack of breeding site fidelity so we did not expect a geographical cline in their biometrics. Sexing criteria available for Little Stints in the literature were developed using museum specimens, which often shrink, leading to misclassification of live birds. The sexing criteria we developed can be used for studies on Little Stints at their non-breeding grounds and on past data, but should be applied cautiously because of the overlapping ranges.
Project description:In artificial insemination the use of sex-sorted bovine sperm results in reduced conception, the causes of which are only partly understood. Therefore, we set out to investigate the effects of sexing on bovine sperm function and early embryonic development. Computer-assisted semen analysis (CASA) of sperm of the same bulls (n?=?5), before and after sexing, demonstrated significantly reduced fast (A) and slow (B) progressively motile sperm (p?<?0.05) after sexing. Sexed-sperm also revealed significantly less hyperactivated sperm (p?<?0.05). As shown by time-lapse videomicroscopy of in vitro produced embryos (n?=?360), embryos derived from sexed-sperm displayed significantly increased incidences of arrest at the 4-cell stage (p?<?0.05). The relative risk for shrinkage/fusion of blastomeres with subsequent lysis was 1.71 times higher in the embryos derived from sexed-sperm as compared to conventional embryos (p?<?0.05) resulting in significantly reduced blastocyst rates (p?<?0.001). The relative risk for cleavage was 2.36 times lower in the embryos derived from sex-sorted sperm (p?<?0.001). Additionally, sexed-sperm-derived embryos showed reduced survival times (hazard ratio HR?=?1.54, p?<?0.001) which were bull dependent (p?<?0.001). However, the percentage of apoptotic cells was similar to conventional embryos. Furthermore, embryos derived from sexed-sperm were found to reach developmental stages at similar timings as conventional embryos. Our results suggest that reduced conception rates after sexing are due to altered sperm morphokinetics, decreasing the chance of sperm to reach and fertilise the oocyte, and aberrant early embryonic development.
Project description:Many bird species are sexually monomorphic and cannot be sexed based on phenotypic traits. Rapid sex determination is often a necessary component of avian studies focusing on behavior, ecology, evolution, and conservation. While PCR-based methods are the most common technique for molecularly sexing birds in the laboratory, a simpler, faster, and cheaper method has emerged, which can be used in the laboratory, but importantly also in the field. Herein, we used loop-mediated isothermal amplification (LAMP) for rapid sex determination of blood samples from juvenile European blackcaps, Sylvia atricapilla, sampled in the wild. We designed LAMP primers unique to S. atricapilla based on the sex chromosome-specific gene, chromo-helicase-DNA-binding protein (CHD), optimized the primers for laboratory and field application, and then used them to test a subset of wild-caught juvenile blackcaps of unknown gender at the time of capture. Sex determination results were fast and accurate. The advantages of this technique are that it allows researchers to identify the sex of individual birds within hours of sampling and eliminates the need for direct access to a laboratory if implemented at a remote field site. This work adds to the increasing list of available LAMP primers for different bird species and is a new addition within the Passeriformes order.
Project description:Background and Aim:Many avian species are considered sexually monomorphic. In monomorphic bird species, especially in young birds, sex is difficult to identify based on an analysis of their external morphology. Accurate sex identification is essential for avian captive breeding and evolutionary studies. Methods with varying degrees of invasiveness such as vent sexing, laparoscopic surgery, steroid sexing, and chromosome inspection (karyotyping) are used for sex identification in monomorphic birds. This study aimed to assess the utility of a non-invasive molecular marker for gender identification in a variety of captive monomorphic birds, as a strategy for conservation. Materials and Methods:DNA was isolated from feather samples from 52 individuals representing 16 species of 11 families indigenous to both Indonesia and elsewhere. We amplified the chromodomain helicase DNA-binding (CHD) gene using polymerase chain reaction with MP, NP, and PF primers to amplify introns with lengths that differ between the CHD-W and the CHD-Z genes, allowing sex discrimination because the W chromosome is exclusively present in females. Results:Molecular bird sexing confirmed 33 females and 19 males with 100% accuracy. We used sequencing followed by alignment on one protected bird species (Probosciger aterrimus). Conclusion:Sex identification may be accomplished noninvasively in birds, because males only have Z sex chromosomes, whereas females have both Z and W chromosomes. Consequently, the presence of a W-unique DNA sequence identifies an individual as female. Sexing of birds is vital for scientific research, and to increase the success rate of conservation breeding programs.
Project description:Abstract Darwin's finches are a classic example of adaptive radiation, a process by which multiple ecologically distinct species rapidly evolve from a single ancestor. Such evolutionary diversification is typically explained by adaptation to new ecological opportunities. However, the ecological diversification of Darwin's finches following their dispersal to Galápagos was not matched on the same archipelago by other lineages of colonizing land birds, which diversified very little in terms of both species number and morphology. To better understand the causes underlying the extraordinary variation in Darwin's finches, we analyze the evolutionary dynamics of speciation and trait diversification in Thraupidae, including Coerebinae (Darwin's finches and relatives) and, their closely related clade, Sporophilinae. For all traits, we observe an early pulse of speciation and morphological diversification followed by prolonged periods of slower steady?state rates of change. The primary exception is the apparent recent increase in diversification rate in Darwin's finches coupled with highly variable beak morphology, a potential key factor explaining this adaptive radiation. Our observations illustrate how the exploitation of ecological opportunity by contrasting means can produce clades with similarly high diversification rate yet strikingly different degrees of ecological and morphological differentiation. The ecological diversification of Darwin's finches is unmatched on the same archipelago by any other clade of colonising land birds. For all morphological traits, we observe an early pulse of diversification followed by prolonged periods of slower steady?state rates of change for the tanager family a substantial increase in speciation rate in Darwin's finches coupled with highly variable beak morphology. Our observations illustrate how the exploitation of ecological opportunity by contrasting means can produce clades high in speciation rates yet strikingly different in their degree of ecological and morphological differentiation.
Project description:Darwin's finches comprise a group of passerine birds first collected by Charles Darwin during his visit to the Galápagos Archipelago. The group, a textbook example of adaptive radiation (the diversification of a founding population into an array of species differentially adapted to diverse environmental niches), encompasses 14 currently recognized species, of which 13 live on the Galápagos Islands and one on the Cocos Island in the Pacific Ocean. Although Darwin's finches have been studied extensively by morphologists, ecologists, and ethologists, their phylogenetic relationships remain uncertain. Here, sequences of two mtDNA segments, the cytochrome b and the control region, have been used to infer the evolutionary history of the group. The data reveal the Darwin's finches to be a monophyletic group with the warbler finch being the species closest to the founding stock, followed by the vegetarian finch, and then by two sister groups, the ground and the tree finches. The Cocos finch is related to the tree finches of the Galápagos Islands. The traditional classification of ground finches into six species and tree finches into five species is not reflected in the molecular data. In these two groups, ancestral polymorphisms have not, as yet, been sorted out among the cross-hybridizing species.
Project description:The extinct Huia (Heteralocha acutirostris) of New Zealand represents the most extreme example of beak dimorphism known in birds. We used a combination of nuclear genotyping methods, molecular sexing, and morphometric analyses of museum specimens collected in the late 19(th) and early 20(th) centuries to quantify the sexual dimorphism and population structure of this extraordinary species. We report that the classical description of Huia as having distinctive sex-linked morphologies is not universally correct. Four Huia, sexed as females had short beaks and, on this basis, were indistinguishable from males. Hence, we suggest it is likely that Huia males and females were indistinguishable as juveniles and that the well-known beak dimorphism is the result of differential beak growth rates in males and females. Furthermore, we tested the prediction that the social organisation and limited powers of flight of Huia resulted in high levels of population genetic structure. Using a suite of microsatellite DNA loci, we report high levels of genetic diversity in Huia, and we detected no significant population genetic structure. In addition, using mitochondrial hypervariable region sequences, and likely mutation rates and generation times, we estimated that the census population size of Huia was moderately high. We conclude that the social organization and limited powers of flight did not result in a highly structured population.
Project description:BACKGROUND:Darwin's finches are a clade of 19 species of passerine birds native to the Galápagos Islands, whose biogeography, specialized beak morphologies, and dietary choices-ranging from seeds to blood-make them a classic example of adaptive radiation. While these iconic birds have been intensely studied, the composition of their gut microbiome and the factors influencing it, including host species, diet, and biogeography, has not yet been explored. RESULTS:We characterized the microbial community associated with 12 species of Darwin's finches using high-throughput 16S rRNA sequencing of fecal samples from 114 individuals across nine islands, including the unusual blood-feeding vampire finch (Geospiza septentrionalis) from Darwin and Wolf Islands. The phylum-level core gut microbiome for Darwin's finches included the Firmicutes, Gammaproteobacteria, and Actinobacteria, with members of the Bacteroidetes at conspicuously low abundance. The gut microbiome was surprisingly well conserved across the diversity of finch species, with one exception-the vampire finch-which harbored bacteria that were either absent or extremely rare in other finches, including Fusobacterium, Cetobacterium, Ureaplasma, Mucispirillum, Campylobacter, and various members of the Clostridia-bacteria known from the guts of carnivorous birds and reptiles. Complementary stable isotope analysis of feathers revealed exceptionally high ?15N isotope values in the vampire finch, resembling top marine predators. The Galápagos archipelago is also known for extreme wet and dry seasons, and we observed a significant seasonal shift in the gut microbial community of five additional finch species sampled during both seasons. CONCLUSIONS:This study demonstrates the overall conservatism of the finch gut microbiome over short (<?1 Ma) divergence timescales, except in the most extreme case of dietary specialization, and elevates the evolutionary importance of seasonal shifts in driving not only species adaptation, but also gut microbiome composition.
Project description:The difficulty in differentiating the sex of monomorphic bird species has made molecular sexing an important tool in addressing this problem. This method uses noninvasively collected materials such as feathers and may be advantageous for sexing endangered as well as commercialized bird species. In this study, seven primer sets for sexing birds were screened in Aerodramus fuciphagus using a total of 13 feather samples that were randomly selected from the state of Perak, Malaysia. From the screening analysis, only one primer set (P8/WZ/W) successfully differentiated the sex of A. fuciphagus. PCR amplification produced a single 255-bp DNA fragment for males which was derived from CHD-Z (CHD gene region in the sex chromosome Z), while for the females it produced two fragments (144 and 255 bp). The 144-bp fragment was from CHD-W (CHD gene region in the sex chromosome W). Results from sequencing showed no variations in the base sequences of the CHD-W and CHD-Z amplified fragments within the same sexes, except for one male sample (A23) where at position 166, a base substitution occurred (G ? A). Phylogenetic analysis of CHD-W showed that four (Apodiformes; Gruiformes; Passeriformes; and Pelecaniformes) out of the five orders investigated had formed four clear clusters within their orders, including the studied order: Apodiformes. Whereas in CHD-Z, four (Accipitriformes; Columbiformes; Galliformes; and Passeriformes) out of five orders investigated formed four clear clusters within their orders, excluding the studied order. In addition, A. fuciphagus and Apus apus (both Apodiformes) showed less divergence in CHD-W than CHD-Z (0% c.f. 9%). The result suggests that in A. fuciphagus, CHD gene evolution occurred at a higher rate in males (CHD-Z) compared to females (CHD-W). This finding may be useful for further studies on sex ratio and breeding management of A. fuciphagus.