Molecular sexing eutherian mammals

Mammals can be molecular sexed by polymerase chain reaction (PCR) amplification of Y chromosome fragments or coamplification of homologous fragments from both sex chromosomes, which are discriminated by size polymorphism or Y‐specific restriction digestion. Although coamplification of X and Y fragments is more reliable, size polymorphism in homologous fragments is uncommon and Y‐specific restriction site identification requires screening with a battery of enzymes or cloning. Here we describe a simple approach, using ‘double peaks’ in the chromatogram upon direct sequencing of PCR products from males, to identify Y‐specific restriction sites, and demonstrate its utility by application to a range of taxa.     

Molecular sexing of African rhinoceros

We report the development of a fast and reliable PCR-based method for sex identification of African Rhinoceros (Ceratotherium simum and Diceros bicornis) that could easily be incorporated into fluorescent short tandem repeat (STR) profiling. A single primer pair, consisting of a fluorescently labelled forward primer and an unlabelled reverse primer, is used to co-amplify homologous fragments of a zinc finger (ZF) protein intron which exhibits size polymorphism between the X and Y chromosomes. In both species, the amplified ZFX and ZFY amplicons differ in size by 7 bp and can thus be differentiated by capillary electrophoresis. Blood, tissue, horn, and faecal samples were correctly sexed using this method. Cross species testing also demonstrated that this method could be used to sex Indian rhinoceros (Rhinoceros unicornis) samples.     

Molecular sexing of birds: A comparative review of polymerase chain reaction (PCR)-based methods

Accurate identification of sex in birds is important for the management and conservation of avian wildlife in several ways, namely in the development of population, behavioral and ecological studies, as well as in the improvement of ex situ captive breeding programs. In general, nestlings, juveniles and adult birds of a wide number of sexually monomorphic species cannot be sexed based on phenotypic traits. The development of molecular methodologies for avian sexing overcame these difficulties, allowing a reliable gender differentiation for these species. The polymerase chain reaction (PCR)-based methods have been widely applied in molecular sexing of birds, using a large diversity of sex-linked markers. During the last 15 yrs, there was a continuous improvement in the PCR-based protocols for bird sexing, increasing the accuracy, speed and high-throughput applicability of these techniques. The recent advances in real-time PCR platforms and whole genome analysis methods provided new resources for the detection and analysis of novel specific markers and protocols. This review presents a comparative guide of classical and recent advances in PCR-based methods for avian molecular sexing, highlighting its strengths and limitations. Future research opportunities in this field are also addressed.     

A triple-primer PCR method for sexing endangered caprine species

Molecular sexing is becoming an essential technique in understanding the sexual structure and dynamics of natural populations. Herein, we report on a triple-primer PCR method based on the last introns of the ZFX/Y alleles for sex identification in Bovidae, and its successful application to five endangered caprine species. The male samples generated a ~230 bp ZFX-specific fragment and a ~140 bp ZFY-specific fragment, and the female samples only generated the ~230 bp fragment. This method is very sensitive to the Y-linked fragment, thus effectively avoiding false negatives. Genomic DNA extracted from well preserved tissues, non-invasive samples and smoked meat are all usable for analysis with this method.     

Molecular sexing and sources of CHD1‐Z/W sequence variation in Hawaiian birds

Sequence information from 28 CHD1 gene fragments reveals that a primary source of variability in CHD1‐W genes is a variable intron microsatellite; a single‐codon deletion was found in the 3′ exon in one species. Sequence variation of CHD1‐Z genes was detected in males that altered polymerase chain reaction (PCR) fragment length. Three sets of CHD1‐based primers were evaluated for sex determination in 12 endemic and 8 alien Hawaiian species, including one of the last po’o‐uli. Combined, these primers provide a reliable means of sex determination in most species (including the po’o‐uli), and have produced a valuable reference database for future expanded population‐level studies.     

Identification of sex-linked SNP markers in wild populations of monomorphic birds

Single-nucleotide polymorphism (SNP) analysis is a powerful tool for population genetics, pedigree reconstruction and phenotypic trait mapping. However, the untapped potential of SNP markers to discriminate the sex of individuals in species with reduced sexual dimorphism or of individuals during immature stages remains a largely unexplored avenue. Here, we developed a novel protocol for molecular sexing of birds based on the detection of unique Z- and W-linked SNP markers. Our method is based on the identification of two unique loci, one in each sexual chromosome. Individuals are considered males when they show no calls for the W-linked SNP and are heterozygous or homozygous for the Z-linked SNP, while females exhibit both Z- and W-linked SNP calls. We validated the method in the Jackdaw (Corvus monedula). The reduced sexual dimorphism in this species makes it difficult to identify the sex of individuals in the wild. We assessed the reliability of the method using 36 individuals of known sex and found that their sex was correctly assigned in 100% of cases. The sex-linked markers also proved to be widely applicable for discriminating males and females from a sample of 927 genotyped individuals at different maturity stages, with an accuracy of 99.5%. Since SNP markers are increasingly used in quantitative genetic analyses of wild populations, the approach we propose has great potential to be integrated into broader genetic research programmes without the need for additional sexing techniques.     

Causes of hatching failure in endangered birds

About 10 per cent of birds'' eggs fail to hatch, but the incidence of failure can be much higher in endangered species. Most studies fail to distinguish between infertility (due to a lack of sperm) and embryo mortality as the cause of hatching failure, yet doing so is crucial in order to understand the underlying problem. Using newly validated techniques to visualize sperm and embryonic tissue, we assessed the fertility status of unhatched eggs of five endangered species, including both wild and captive birds. All eggs were classified as ‘infertile’ when collected, but most were actually fertile with numerous sperm on the ovum. Eggs of captive birds had fewer sperm and were more likely to be infertile than those of wild birds. Our findings raise important questions regarding the management of captive breeding programmes.     

一种简单通用的鸟类性别分子鉴定技术（简报）

我国幅员辽阔,生物类型多种多样,是世界上拥有鸟类种类最多的国家之一。截至1999年底,已知有鸟类1253种948亚种,隶属于21目83科,其中有多种为我国特有或珍稀濒危的鸟类。近年来由于环境污染、植被破坏及非法捕猎等种种原因,许多鸟类尤其是珍稀鸟类正逐步濒临灭绝。为了加紧     

Improving the reliability of molecular sexing of birds using a W-specific marker

Molecular techniques for identifying sex of birds utilize length differences between CHD-Z and CHD-W introns, but in some cases these methods can lead to sexing errors. Here we show that an additional W-specific primer can be used in conjunction with a pre-existing sexing primer pair to dramatically improve the reliability of molecular sexing methods. We illustrate the approach with American coots (Fulica americana), a species with CHD-Z polymorphism that could not be accurately sexed using traditional methods. We developed a reverse primer GWR2 designed to sit within the intron of the W chromosome and amplify a distinctively small DNA fragment that serves as a W-specific marker. Analysis of known-sex individuals indicates that this W-specific primer provides an efficient and reliable protocol to identify the sex of F. americana. The development of such sex-specific primers will likely increase the reliability of molecular sexing methods in other birds as well. Comparisons between CHD-Z alleles of coots and common moorhens (Gallinula chloropus) revealed that CHD-Z polymorphism evolved separately in these two closely related species. We discuss the implications of repeated evolution of CHD-Z polymorphisms among birds.     

The conservation of critically endangered flightless birds in New Zealand

The current status of Kakapo Strigops habroptilus and Takahe Porphyrio mantelli is described along with recent developments in programmes for their conservation. Both species were (at different times) thought to be effectively extinct, and both have been temporarily reprieved by the discovery of new populations. Population declines have continued, with Kakapo now reduced to less than 50 individuals and Takahe to about 150. Kakapo are especially at risk; 87% of the remaining birds are over 14 years old and only 17 females are known. Research on relict populations of both species has identified predation and competition from introduced mammals as major threats. Both species have high rates of egg infertility and low survival of young. Increasingly intensive management of both Kakapo and Takahe over recent years has included translocation to predator-free island refuges, supplementary feeding to encourage breeding, clutch manipulation, captive rearing and predator control. All known Kakapo have now been transferred to three island refuges, where the overall rate of population decline has slowed and supplementary feeding has apparently encouraged more frequent breeding attempts. Takahe conservation has concentrated largely on attempts to increase the population in Fiordland, New Zealand, through clutch manipulation and release of captive-reared young, but birds have also been released on four islands, which now hold 19% of the total population. The relict Fiordland populations of both Kakapo and Takahe were confined to apparently suboptimal habitat. Both species have successfully adapted to novel environments and foods when translocated, and the populations which now exist present improved opportunities for intensive management using a range of conservation techniques to enhance productivity and survival. Recent population trends of Kakapo and Takahe are reconstructed, and the contribution of research to their conservation is reviewed.     

Molecular evolutionary genomics of birds

Insight into the molecular evolution of birds has been offered by the steady accumulation of avian DNA sequence data, recently culminating in the first draft sequence of an avian genome, that of chicken. By studying avian molecular evolution we can learn about adaptations and phenotypic evolution in birds, and also gain an understanding of the similarities and differences between mammalian and avian genomes. In both these lineages, there is pronounced isochore structure with highly variable GC content. However, while mammalian isochores are decaying, they are maintained in the chicken lineage, which is consistent with a biased gene conversion model where the high and variable recombination rate of birds reinforces heterogeneity in GC. In Galliformes, GC is positively correlated with the rate of nucleotide substitution; the mean neutral mutation rate is 0.12-0.15% at each site per million years but this estimate comes with significant local variation in the rate of mutation. Comparative genomics reveals lower d(N)/d(S) ratios on micro- compared to macrochromosomes, possibly due to population genetic effects or a non-random distribution of genes with respect to chromosome size. A non-random genomic distribution is shown by genes with sex-biased expression, with male-biased genes over-represented and female-biased genes under-represented on the Z chromosome. A strong effect of selection is evident on the non-recombining W chromosome with high d(N)/d(S) ratios and limited polymorphism. Nucleotide diversity in chicken is estimated at 4-5 x 10(-3) which might be seen as surprisingly high given presumed bottlenecks during domestication, but is lower than that recently observed in several natural populations of other species. Several important aspects of the molecular evolutionary process of birds remain to be understood and it can be anticipated that the upcoming genome sequence of a second bird species, the zebra finch, as well as the integration of data on gene expression, shall further advance our knowledge of avian evolution.     

An endangered bird calls less when invasive birds are calling

Novel noises can affect various animal behaviours, and changes to vocal behaviour are some of the most documented. The calls of invasive species are an important source of novel noise, yet their effects on native species are poorly understood. We examined the effects of invasive bird calls on the vocal activity of an endangered Australian finch to investigate whether: 1) native finch calling behaviour was affected by novel invasive bird calls, and 2) the calls of the finches overlapped in frequency with those of invasive birds. We exposed a wild population of black‐throated finch southern subspecies Poephila cincta cincta to the vocalisations of two invasive birds, nutmeg mannikins Lonchura punctulata and common mynas Acridotheres tristis, a synthetic ‘pink' noise, and a silent control. To determine whether the amount of black‐throated finch calling differed in response to treatments, we recorded and quantified black‐throated finch vocalisations, and assessed the amount of calling using a generalised linear mixed model followed by pairwise comparisons. We also measured, for both black‐throated finches and the stimulus noises: dominant, minimum and maximum frequency, and assessed the degree of frequency overlap between black‐throated finch calls and stimulus noises. Compared to silent controls, black‐throated finches called less when exposed to common myna calls and pink noise, but not to nutmeg mannikin calls. We also found that pink noise overlapped most in frequency with black‐throated finch calls. Common myna calls also somewhat overlapped the frequency range of black‐throated finch calls, whereas nutmeg mannikin calls overlapped the least. It is possible that masking interference is the mechanism behind the reduction in calling in response to common myna calls and pink noise, but more work is needed to resolve this. Regardless, these results indicate that the calls of invasive species can affect the behaviour of native species, and future research should aim to understand the scope and severity of this issue.     

Molecular sexing in the Mediterranean fruit fly, Ceratitis capitata

Molecular methods have been devised for sexing Mediterranean fruit fly (medfly) individuals using minimal amounts of material from any stage of the life cycle. Molecular sexing methods are particularly valuable when material is obtained from pre-adult stages and sex identification based on morphological characters is not possible. These methods may also be useful for adult stage material in situations where only limited amounts or poorly preserved specimens are available. The sexing methods described here use the polymerase chain reaction (PCR) to amplify sequences known to originate from the sex chromosomes of this species. One method co-amplifies homologous regions of the ITS1 ribosomal DNA from both the X and Y chromosomes. Males and females are distinguished based on the restriction fragment pattern produced after digestion of the PCR products with the restriction enzyme ApoI. A second method identifies males based on the positive amplification of a repetitive DNA sequence originating from the Y chromosome. Both methods are shown to be capable of establishing the sex identity of individuals using only minimal amounts of material from any stage of the life cycle.     

A triple-primer PCR approach for the sex identification of endangered Phasianidae birds

To manage and conserve endangered Phasianidae species and to study their ecology, behavior, and population structure, sex identification is necessary. A search for the pheasant sex-linked marker (PSL) was carried out by comparing the sequences of chicken sex chromosomes. A triple-primer PCR approach was established in the present study based on a novel PSL for the sex identification of Phasianidae birds. The triple-primer system amplified a 202-bp PSL-W-specific fragment in female Phasianidae birds, whereas there is a 107-bp PSL-Z-specific fragment in the both sexes, thus making the female distinct. We also found a 102-bp AT-rich repeat sequence inserted into the PSL-W-specific region in Tetraophasis szechenyii generating a ∼300-bp PSL-W fragment in females. The sex of 11 Phasianidae species was distinguished clearly by the triple-primer PCR approach indicating that this method is both quick and reliable and can be easily applied to a wider range of Phasianidae species. Due to the relatively small size of the amplification products, the triple-primer PCR approach can also be applied to noninvasive samples such as feathers.     

Tourism as a threat to critically endangered and endangered birds: global patterns and trends in conservation hotspots

More than 12 % of bird species are threatened with extinction. Numerous anthropogenic activities and processes are considered responsible for such declines, including tourism related activities. These activities often occur in global biodiversity hotspots but few studies consider the potential risks associated with tourism. The relative importance of tourism as a threat to birds was quantified using a global analysis of the threats facing critically endangered and endangered birds in the hotspots. Sixty-three critically endangered and endangered bird species are reportedly threatened by tourism. Among those 63 species, marine, coastal and aquatic birds are threatened more by tourism than was expected. Hotspots with the most species threatened by tourism are Polynesia–Micronesia and the Mediterranean Basin. This study uses individual threatening processes in a new way to characterise hotspots for conservation action, advancing previous identification criteria. Analysing hotspots in terms of the relative presence of individual threatening processes may help to more effectively direct future research in these priority regions.     

Molecular sexing of the communally breeding pukeko: an important ecological tool

A central biological parameter in the study of any animal population is the accurate assignment of sex. Indeed any ecological study of a population requires information on sex composition in relation to such biological factors as behaviour, movement, mortality and birth rate. However, our ability to assign the sex of adults of many avian species is poor and the sexing of young is universally difficult. We report here the successful application of a molecular technique for the assignment of sex in the communally breeding pukeko or purple swamphen ( Porphyrio porphyrio melanotus ). W- and Z-linked chromosome fragments in digested genomic DNA of pukeko were detected with the DNA probe pMg1. We consequently show that this species breeds in polyandrous, polygynous and polygynandrous groups. Finally we discuss why recent molecular methods represent important new tools in ecology.