New and improved molecular sexing methods for museum bird specimens

We present two new avian molecular sexing techniques for nonpasserine and passerine birds (Neognathae), which are more suitable for use with museum specimens than earlier methods. The technique for nonpasserines is based on a new primer (M5) which, in combination with the existing P8 primer, targets a smaller amplicon in the CHD1 sex-linked gene than previously. Primers targeting ATP5A1, an avian sex-linked gene not previously used for sex identification, were developed for passerines. Comprehensive testing across species demonstrated that both primer pairs sex a range of different species within their respective taxonomic groups. Rigorous evaluation of each method within species showed that these permitted sexing of specimens dating from the 1850s. For corn bunting museum specimens, the ATP5A1 method sexed 98% of 63 samples (1857-1966). The M5/P8 CHD1 method was similarly successful, sexing 90% of 384 moorhen specimens from six different museum collections (1855-2001). In contrast, the original P2/P8 CHD1 sexing method only identified the sex of less than half of 111 museum moorhen samples. In addition to dried skin samples, these methods may be useful for other types of material that yield degraded or damaged DNA, and are hence potential new sexing tools for avian conservation genetics, population management and wildlife forensics.     

Sex identification by alternative polymerase chain reaction methods in falconiformes

A number of avian species are difficult to sex morphologically, especially as nestlings. Like other avian species, many species of Falconiformes are sexually monomorphic. Therefore, it is desirable that new methods based on DNA analysis are established in Falconiformes and other sexual monomorphic species. We identified sex in Falconiformes by two alternative methods. First, we used a sexing method based on the intronic length variation between CHD1W and CHD1Z using primers flanking the intron. In this method, two species of Falconidae could be identified for sexing. However, six species of Accipitridae could not, because they have few length variations. The second method used was based on differences in sequences between CHD1W and CHD1Z. From sequence analysis, a 3'-terminal mismatch primer on point mutation conserved among Falconiformes was designed, and identification of sex with the amplification refractory mutation system (ARMS) was performed. This method could identify sex in all species tested. In addition, because the 3'-terminal mismatch primer was designed on a point mutation conserved among Falconiformes, ARMS with these primers may identify sex in all Falconiformes. These are simple and rapid sexing methods, since only polymerase chain reaction (PCR) and agarose electrophoresis are required. In conclusion, sex identification by an alternative PCR approach based on intronic length variation and on differences in sequences between CHD1W and CHD1Z proved applicable to and useful for Falconiformes.     

Flow cytometric sexing of mammalian sperm

This review reexamines parameters needed for optimization of flow cytometric sexing mammalian sperm and updates the current status of sperm sexing for various species where this technology is currently being applied. Differences in DNA content have provided both a method to differentiate between these sex-determining gametes and a method to sort them that can be used for predetermining sex in mammals. Although the DNA content of all cells for each mammalian species is highly conserved, slight but measurable DNA content differences of sperm occur within species even among cattle breeds due to different sizes of Y-chromosomes. Most mammals produce flattened, oval-headed sperm that can be oriented within a sorter using hydrodynamic forces. Multiplying the percentage the difference in DNA content of the X- or Y-chromosome bearing sperm times the area of the flat profile of the sperm head gives a simple sorting index that suggests that bull and boar sperm are well suited for separation in a flow sorter. Successful sperm sexing of various species must take into account the relative susceptibilities of gametes to the stresses that occur during sexing. Sorting conditions must be optimized for each species to achieve acceptable sperm sexing efficiency, usually at 90% accuracy. In the commercial application of sperm sexing to cattle, fertility of sex-sorted bull sperm at 2 x 10(6)/dose remains at 70-80% of unsexed sperm at normal doses of 10 to 20 x 10(6) sperm. DNA content measurements have been used to identify the sex-chromosome bearing sperm populations with good accuracy in semen from at least 23 mammalian species, and normal-appearing offspring have been produced from sexed sperm of at least seven species.     

Molecular sexing of monomorphic endangered Ara birds

Survival of most endangered birds may depend on breeding programs where sex identification plays an important role. Molecular sexing has shown to be a rapid and safe procedure. In this work we established sex identification of monomorphic endangered Ara birds using a chromosome W-linked DNA marker, the Chromo-helicase-DNA-Binding 1 (CHD) gene. Most birds have two CHD sex-linked genes, one W-linked (CHD-W) and one Z-linked (CHD-Z). These markers were characterized from Ara militaris and gender sex was determined by PCR and restriction analyzes. The procedure here reported was successfully applied to five different species of the genus Ara and confirmed the validity of the technique. To our knowledge, this is the first report of molecular sexing of the Ara species. This molecular sexing is currently been used in breeding programs of Ara birds.     

Biometric sex discrimination is unreliable when sexual dimorphism varies within and between years: an example in Eurasian Oystercatchers Haematopus ostralegus

Molecular sexing of birds has been possible for over a decade, but for practical reasons many studies still use biometric data for sex discrimination. In some species, the sexes are easy to distinguish but sexual dimorphism is often more subtle, requiring the use of statistical analyses of biometric measurements to discriminate sexes. These models are usually parameterized and validated using data from a limited number of sites and years. However, the resulting discriminant functions are often applied to other populations and periods. A crucial, but usually untested, assumption is that sexual dimorphism does not vary in time and space. Here we illustrate the consequences of violation of this assumption in Eurasian Oystercatchers Haematopus ostralegus , a species for which most studies have relied on biometric sexing. Using biometric data from captures of known-sex birds, we show that sexual dimorphism varied substantially in time and even reversed in some months and years. Furthermore, some biometric traits used in sexing changed gradually over time, causing a reduction in sexual dimorphism. We show that the consequences of this variation on sex discrimination in Oystercatchers are subtle and easily overlooked, but can result in inaccurate and strongly male- or female-biased sex-ratio estimates. We recommend that biometric sexing should be avoided in Oystercatchers unless specific calibration for each month, year and area is carried out. This recommendation also applies to other species where biometric traits may depend on environmental conditions. We argue that this condition might apply to many bird species and therefore advise caution when interpreting results based on biometric sex discrimination.     

Multiple molecular approaches yield no evidence for sex‐determining genes in lake sturgeon (Acipenser fulvescens)

Common DNA‐based sexing assays have been widely used for the conservation and management of mammals and birds. However, many fishes do not have genetic sex determination and in those that do, the plasticity of the genes involved means that species‐specific assays are normally required. Such DNA‐sexing markers would be especially valuable in lake sturgeon (Acipenser fulvescens) because of their sexual monomorphism, delayed sexual maturity, and conservation status. We tried to identify genetic differences between male and female lake sturgeon using several different molecular genetic methods, including randomly amplified polymorphic DNA, representational difference analyses, subtractive hybridization, and a candidate gene approach. Ultimately, a number of genes were identified but none was sex‐specific. Although the ultimate mechanism of sex determination is yet unknown, it is possible that sex determination is environmental in lake sturgeon, especially since recent studies have also failed to identify sex determination genes in other sturgeon species.     

Development of genetic sexing strains in Lepidoptera: from traditional to transgenic approaches

The sterile insect technique (SIT) is currently being used for the control of many agricultural pests, including some lepidopteran species. The SIT relies on the rearing and release of large numbers of genetically sterile insects into a wild population. The holokinetic chromosomes of Lepidoptera respond differently to radiation than do species where there is a localized centromere. This difference has enabled a variation of the SIT to be developed for Lepidoptera where a substerilizing dose of radiation is given to the insects before their release with the result that a certain level of sterility is inherited by the F1 offspring. The development of genetic sexing strains for fruit flies, enabling the release of males only, has resulted in enormous economic benefits in the mass rearing and has increased the efficiency of the field operations severalfold. This article outlines Mendelian approaches that are currently available to separate large numbers of males and females efficiently for different lepidopteran species and describes their difficulties and constraints. Successful transgenesis in several lepidopteran species opens up new possibilities to develop genetic sexing strains. The proposal to develop genetic sexing strains described in this article takes advantage of the fact that in Lepidoptera, the female is the heterogametic sex, with most species having aWZ sex chromosome pair, whereas the males are ZZ. This means that if a conditional lethal gene can be inserted into the W chromosome, then all females should die after the application of the restrictive condition. The assumptions made to accommodate this model are discussed, and the advantages to be gained for control programs are elucidated.     

Morphology versus molecules: sexing Red-winged Blackbird nestlings

ABSTRACT.   Past studies of offspring sex ratios in birds have often relied on sexually size dimorphic species where nestling sex could be determined based on weight at a given age. DNA-based sexing techniques allow us to assess the accuracy of those techniques and to refine them for use when costs or convenience make DNA methods impractical. Using nestling Red-winged Blackbirds ( Agelaius phoeniceus ) whose sex was determined using DNA, we compared sex ratios obtained using different morphological criteria. Conservative criteria from previous studies were completely accurate, but allowed sexing of few nestlings younger than 8 d old, and were more successful for sexing males than females. A new method was developed that allowed accurate sexing of nestlings beginning at day 6 posthatching and was less biased relative to known sex ratios. Using 11 years of data, the conservative method left an average of 55% of nestlings and 36% of fledglings unsexed, compared to 31% and 9% using the new method. Furthermore, the male bias in sex ratio estimates using the conservative method was greater, both absolutely and relative to estimates based on the new method, when the proportion of unsexed nestlings (because they were not weighed when older) was higher. Thus, estimates of population sex ratios will be more accurate as the number of nestlings measured on day 8 or older increases. However, if some nestlings that were not weighed past day 7 fledge, the new method allows more of those individuals to be sexed than the conservative method, and the population sex ratio estimate should be more reliable. Although our approach should apply to other sexually dimorphic species, the criteria used must be developed based on such species-specific attributes as growth patterns and degree of hatching asynchrony.     

PCR-based sexing in conservation biology: Wrong answers from an accurate methodology?

Molecular tests of sex based on the polymerase chain reaction (PCR) are now commonplace in conservation biology, routinely guiding management decisions. While molecular approaches to sexing can be highly reliable, current practices may leave an undesirable level of uncertainty in the sexes identified, because researchers focus on determining the sex-specific nature of a test, largely ignoring the accuracy of the test to correctly sex individuals. This latter step requires considerably more known-sex individuals. We argue that, due to the well-known technical problems associated with PCR amplification, the demonstrated potential for sexing errors and few known-sex individuals being available from threatened species, conservationists should place greater emphasis on verifying the sexes identified with PCR tests. We propose that all individuals of the sex indistinguishable from an amplification failure (e.g., females in mammals XX, males in birds ZZ) should be verified with a second independent sex test. Such a consensus approach to molecular sexing would reduce errors that could arise due to technical failure and PCR anomalies, but may also reduce field and laboratory bookkeeping errors.     

Sex ratios observed in 80 species of parrots

A number of potential evolutionary and physiological factors may be involved in avian sex ratio bias so that under certain conditions a sex ratio bias may favour males or females within a population. In addition different factors may be important in manipulating sex ratio bias through the different life stages. In this study sex ratio bias was examined in a total of 16 570 captive parrots, representing 80 species, many of which are endangered in the wild, using database records originating form commercial laboratories that offer genetic sexing. Within the species examined 72% showed a male bias this was significant in three species, when adjusted for multiple comparisons. This preliminary study is limited due to lack of data on the age of the individuals sampled. However, the large dataset do suggest that this phenomenon should be further considered by investigators working at a species level where such data can be collected.     

Noninvasive molecular sexing: An evaluation and validation of the SRY‐ and amelogenin‐based method in three new lemur species

Many lemur species are arboreal, elusive, and/or nocturnal and are consequently difficult to approach, observe and catch. In addition, most of them are endangered. For these reasons, non‐invasive sampling is especially useful in primates including lemurs. A key issue in conservation and ecological studies is to identify the sex of the sampled individuals to investigate sex‐biased dispersal, parentage, social organization and population sex ratio. Several molecular tests of sex are available in apes and monkeys, but only a handful of them work in the lemuriform clade. Among these tests, the coamplification of the SRY gene with the amelogenin X gene using strepsirhine‐specific X primers seems particularly promising, but the reliability and validity of this sexing test have not been properly assessed yet. In this study, we (i) show that this molecular sexing test works on three additional lemur species (Microcebus tavaratra, Propithecus coronatus and P. verreauxi) from two previously untested genera and one previously untested family, suggesting that these markers are likely to be universal among lemurs and other strepsirrhines; (ii) provide the first evidence that this PCR‐based sexing test works on degraded DNA obtained from noninvasive samples; (iii) validate the approach using a large number of known‐sex individuals and a multiple‐tubes approach, and show that mismatches between the field sex and the final molecular consensus sex occur in less than 10% of all the samples and that most of these mismatches were likely linked to incorrect sex determinations in the field rather than genotyping errors. Am J Phys Anthropol, 2013. © 2013 Wiley Periodicals, Inc.     

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.     

Sex identification of owls (family Strigidae) using oligonucleotide microarrays

Molecular sexing of the diversified avian family Strigidae is difficult. Sex identification using the intron length difference between W and Z chromosomal CHD1 genes, as visualized by agarose gel electrophoreses, often produces ambiguous results. Here we describe a simple method for sexing a variety of Strigidae species using oligonucleotide microarrays, on which several sex-specific probes operated complementarily or in concert. The sex of 8 owl species was identified clearly on the microarrays through sequence recognition. This sequence-directed method can be easily applied to a wider range of Strigidae species.     

Using genotyping data to assign markers to their chromosome type and to infer the sex of individuals: a Bayesian model‐based classifier

The recent democratization of next‐generation‐sequencing‐based approaches towards nonmodel species has made it cost‐effective to produce large genotyping data sets for a wider range of species. However, when no detailed genome assembly is available, poor knowledge about the organization of the markers within the genome might hamper the optimal use of this abundant information. At the most basic level of genomic organization, the type of chromosome (autosomes, sex chromosomes, mitochondria or chloroplast in plants) may remain unknown for most markers which might be limiting or even misleading in some applications, particularly in population genetics. Conversely, the characterization of sex‐linked markers allows molecular sexing of the individuals. In this study, we propose a Bayesian model‐based classifier named detsex, to assign markers to their chromosome type and/or to perform sexing of individuals based on genotyping data. The performance of detsex is further evaluated by a comprehensive simulation study and by the analysis of real data sets from various origins (microsatellite and SNP data derived from genotyping assay designs and NGS experiments). Irrespective of the origin of the markers or the size of the data set, detsex was proved efficient (i) to identify the sex‐linked markers, (ii) to perform molecular sexing of the individuals and (iii) to perform basic quality check of the genotyping data sets. The underlying structure of the model also allows to consider each of these potential applications either separately or jointly.     

Poorly differentiated XX/XY sex chromosomes are widely shared across skink radiation

Differentiated sex chromosomes are believed to be evolutionarily stable, while poorly differentiated sex chromosomes are considered to be prone to turnovers. With around 1700 currently known species forming ca 15% of reptile species diversity, skinks (family Scincidae) are a very diverse group of squamates known for their large ecological and morphological variability. Skinks generally have poorly differentiated and cytogenetically indistinguishable sex chromosomes, and their sex determination was suggested to be highly variable. Here, we determined X-linked genes in the common sandfish (Scincus scincus) and demonstrate that skinks have shared the same homologous XX/XY sex chromosomes across their wide phylogenetic spectrum for at least 85 million years, approaching the age of the highly differentiated ZZ/ZW sex chromosomes of birds and advanced snakes. Skinks thus demonstrate that even poorly differentiated sex chromosomes can be evolutionarily stable. The conservation of sex chromosomes across skinks allows us to introduce the first molecular sexing method widely applicable in this group.     

Molecular sexing assays in 114 mammalian species: In silico sequence reanalysis and a unified graphical visualization of diagnostic tests

Molecular‐based methods for identifying sex in mammals have a wide range of applications, from embryo manipulation to ecological studies. Various sex‐specific or homologous genes can be used for this purpose, PCR amplification being a common method. Over the years, the number of reported tests and the range of tested species have increased greatly. The aim of the present analysis was to retrieve PCR‐based sexing assays for a range of mammalian species, gathering the gene sequences from either the articles or online databases, and visualize the molecular design in a uniform manner. For nucleotide alignment and diagnostic test visualization, the following genomic databases and tools were used: NCBI, Ensembl Nucleotide BLAST, ClustalW2, and NEBcutter V2.0. In the 45 gathered articles, 59 different diagnostic tests based on eight different PCR‐based methods were developed for 114 mammalian species. Most commonly used genes for the analysis were ZFX, ZFY, AMELX, and AMELY. The tests were most commonly based on sex‐specific insertions and deletions (SSIndels) and sex‐specific sequence polymorphisms (SSSP). This review provides an overview of PCR‐based sexing methods developed for mammals. This information will facilitate more efficient development of novel molecular sexing assays and reuse of previously developed tests. Development of many novel and improvement of previously developed tests is also expected with the rapid increase in the quantity and quality of available genetic information.     

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.     

Species and sex identification of the Korean goral (Nemorhaedus caudatus) by molecular analysis of non-invasive samples

Korean long-tailed goral (Nemorhaedus caudatus) is one of the most endangered species in South Korea. However, detailed species distribution and sex ratio data on the elusive goral are still lacking due to difficulty of identification of the species and sex in the field. The primary aim of this study was to develop an economical PCR-RFLP method to identify species using invasive or non-invasive samples from five Korean ungulates: goral (N. caudatus), roe deer (Capreolus pygargus), feral goat (Capra hircus), water deer (Hydropotes inermis) and musk deer (Moschus moschiferus). The secondary aim was to find more efficient molecular sexing techniques that may be applied to invasive or non-invasive samples of ungulate species. We successfully utilized PCR-RFLP of partial mitochondrial cytochrome b gene (376 bp) for species identification, and sex-specific amplification of ZFX/Y and AMELX/Y genes for sexing. Three species (goral, goat and water deer) showed distinctive band patterns by using three restriction enzymes (XbaI, StuI or SspI). Three different sexing primer sets (LGL331/335 for ZFX/Y gene; SE47/48 or SE47/53 for AMELX/Y gene) produced sex-specific band patterns in goral, goat and roe deer. Our results suggest that the molecular analyses of non-invasive samples might provide us with potential tools for the further genetic and ecological study of Korean goral and related species.     

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.