A DNA test to sex most birds

Birds are difficult to sex. Nestlings rarely show sex-linked morphology and we estimate that adult females appear identical to males in over 50% of the world's bird species. This problem can hinder both evolutionary studies and human-assisted breeding of birds. DNA-based sex identification provides a solution. We describe a test based on two conserved CHD (chromo-helicase-DNA-binding) genes that are located on the avian sex chromosomes of all birds, with the possible exception of the ratites (ostriches, etc.; Struthioniformes). The CHD-W gene is located on the W chromosome; therefore it is unique to females. The other gene, CHD-Z, is found on the Z chromosome and therefore occurs in both sexes (female, ZW; male, ZZ). The test employs PCR with a single set of primers. It amplifies homologous sections of both genes and incorporates introns whose lengths usually differ. When examined on a gel there is a single CHD-Z band in males but females have a second, distinctive CHD-W band.     

基于CHD基因序列的18种猛禽鸟类系统发育关系

对18个猛禽CHD基因的一段内含子序列进行比较和分析.CHD-W和CHD-Z基因的多态性存在差异,CHD-W基因不适合种间系统发生学的研究.通过对CHD-Z基因扩增序列构建的NJ和ML树显示:隼科与其他猛禽物种关系较远;鹰科鸟类与鸮形目鸟类亲缘关系较近;在白腹鹞的分类地位上与传统形态学分类不一致;长耳鸮、领角鸮、花彩角鸮、西部鸣角鸮的分类地位存在分歧.鸮形目和隼形目鸟类的CHD-W基因大小有明显区别,支持形态学分类结果,与CHD-Z序列分析结果明显不同.     

Retroposon insertions and the chronology of avian sex chromosome evolution

The vast majority of extant birds possess highly differentiated Z and W sex chromosomes. Nucleotide sequence data from gametologs (homologs on opposite sex chromosomes) suggest that this divergence occurred throughout early bird evolution via stepwise cessation of recombination between identical sex chromosomal regions. Here, we investigated avian sex chromosome differentiation from a novel perspective, using retroposon insertions and random insertions/deletions for the reconstruction of gametologous gene trees. Our data confirm that the CHD1Z/CHD1W genes differentiated in the ancestor of the neognaths, whereas the NIPBLZ/NIPBLW genes diverged in the neoavian ancestor and independently within Galloanserae. The divergence of the ATP5A1Z/ATP5A1W genes in galloanserans occurred independently in the chicken, the screamer, and the ancestor of duck-related birds. In Neoaves, this gene pair differentiated in each of the six sampled representatives, respectively. Additionally, three of our investigated loci can be utilized as universal, easy-to-use independent tools for molecular sexing of Neoaves or Neognathae.     

Molecular sexing of unusually large numbers of Spheniscus magellanicus (Spheniscidae) washed ashore along the Brazilian coast in 2008

There have been few studies on Magellanic penguins (Spheniscus magellanicus). In 2008, these penguins washed ashore along the Brazilian coast in unusually high numbers, some reaching as far as northeast Brazil. As Magellanic penguins show little sexual dimorphism, sex determination by morphological features is not accurate. Here, we tested a molecular procedure for sexing specimens of S. magellanicus washed ashore along the coasts of Sergipe, Rio de Janeiro and Rio Grande do Sul in 2008, comparing the sex ratio between these localities. Tissue samples were collected from 135 dead, beached specimens. We carried out total genomic DNA extraction and CHD-Z/CHD-W gene amplification by PCR using P2 and P8 primers. Amplicons were separated by 12% acrylamide gel electrophoresis. We found a greater proportion of females (70%). Sex could be determined because females have two intronic regions of CHD gene of different size in the sex chromosomes, visualized as two bands on the gel (380 and 400 bp approximately), while males have only one (400 bp). Therefore, this method proved to be effective and sensitive for sex determination of S. magellanicus individuals. Data on sex ratios are useful for understanding the dynamics and ecology of Magellanic penguin populations.     

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.     

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.     

Contrasting levels of nucleotide diversity on the avian Z and W sex chromosomes

Sex chromosomes may provide a context for studying the local effects of mutation rate on molecular evolution, since the two types of sex chromosomes are generally exposed to different mutational environments in male and female germ lines. Importantly, recent studies of some vertebrates have provided evidence for a higher mutation rate among males than among females. Thus, in birds, the Z chromosome, which spends two thirds of its time in the male germ line, is exposed to more mutations than the female-specific W chromosome. We show here that levels of nucleotide diversity are drastically higher on the avian Z chromosome than in paralogous sequences on the W chromosome. In fact, no intraspecific polymorphism whatsoever was seen in about 3.4 kb of CHD1W intron sequence from a total of >150 W chromosome copies of seven different bird species. In contrast, the amount of genetic variability in paralogous sequences on the Z chromosome was significant, with an average pairwise nucleotide diversity (d) of 0.0020 between CHD1Z introns and with 37 segregating sites in a total of 3.8 kb of Z sequence. The contrasting levels of genetic variability on the avian sex chromosomes are thus in a direction predicted from a male-biased mutation rate. However, although a low gene number, as well as some other factors, argues against background selection and/or selective sweeps shaping the genetic variability of the avian W chromosome, we cannot completely exclude selection as a contributor to the low levels of variation on the W chromosome.     

The multiple sex chromosomes of platypus and echidna are not completely identical and several share homology with the avian Z

Background

Sex-determining systems have evolved independently in vertebrates. Placental mammals and marsupials have an XY system, birds have a ZW system. Reptiles and amphibians have different systems, including temperature-dependent sex determination, and XY and ZW systems that differ in origin from birds and placental mammals. Monotremes diverged early in mammalian evolution, just after the mammalian clade diverged from the sauropsid clade. Our previous studies showed that male platypus has five X and five Y chromosomes, no SRY, and DMRT1 on an X chromosome. In order to investigate monotreme sex chromosome evolution, we performed a comparative study of platypus and echidna by chromosome painting and comparative gene mapping.

Results

Chromosome painting reveals a meiotic chain of nine sex chromosomes in the male echidna and establishes their order in the chain. Two of those differ from those in the platypus, three of the platypus sex chromosomes differ from those of the echidna and the order of several chromosomes is rearranged. Comparative gene mapping shows that, in addition to bird autosome regions, regions of bird Z chromosomes are homologous to regions in four platypus X chromosomes, that is, X₁, X₂, X₃, X₅, and in chromosome Y₁.

Conclusion

Monotreme sex chromosomes are easiest to explain on the hypothesis that autosomes were added sequentially to the translocation chain, with the final additions after platypus and echidna divergence. Genome sequencing and contig anchoring show no homology yet between platypus and therian Xs; thus, monotremes have a unique XY sex chromosome system that shares some homology with the avian Z.     

The complete sequence of the mitochondrial genome of Buteo buteo (Aves, Accipitridae) indicates an early split in the phylogeny of raptors

The complete sequence of the mitochondrial (mt) genome of Buteo buteo was determined. Its gene content and nucleotide composition are typical for avian genomes. Due to expanded noncoding sequences, Buteo possesses the longest mt genome sequenced so far (18,674 bp). The gene order comprising the control region and neighboring genes is identical to that of Falco peregrinus, suggesting that the corresponding rearrangement occurred before the falconid/accipitrid split. Phylogenetic analyses performed with the mt sequence of Buteo and nine other mt genomes suggest that for investigations at higher taxonomic levels (e.g., avian orders), concatenated rRNA and tRNA gene sequences are more informative than protein gene sequences with respect to resolution and bootstrap support. Phylogenetic analyses indicate an early split between Accipitridae and Falconidae, which, according to molecular dating of other avian divergence times, can be assumed to have taken place in the late Cretaceous 65-83 MYA.     

Sexing a wider range of avian species based on two CHD1 introns with a unified reaction condition

Identifying the sex of a bird is important to ensure successful breeding strategies and effective conservation programs. Sex may be identified from the intron size of the CHD1 gene located on the avian sex chromosomes Z and W. However, because of the great nucleotide diversity across different avian species, no given intron is in widespread use without ambiguous results. Complicated modifications of the reaction condition are required to suit different species. Two CHD1 introns were used with a unified reaction condition in this study to simplify the procedure. Consequently, genders of 73 avian species covering 19 families were successfully identified based on this two‐intron approach. This means the ability to sex a wider range of avian species using a simplified procedure, greatly assisting in population management at zoos. Zoo Biol 26:425–431, 2007. © 2007 Wiley‐Liss, Inc.     

Increased differentiation and reduced gene flow in sex chromosomes relative to autosomes between lineages of the brown creeper Certhia americana

The properties of sex chromosomes, including patterns of inheritance, reduced levels of recombination, and hemizygosity in one of the sexes may result in the faster fixation of new mutations via drift and natural selection. Due to these patterns and processes, the two rules of speciation to describe the genetics of postzygotic isolation, Haldane's rule and the large‐X effect, both explicitly include quicker evolution on sex chromosomes relative to autosomes. Because sex‐linked mutations may be the first to become fixed in the speciation process, and appear to be due to stronger genetic drift (in birds), we may identify pronounced genetic differentiation in sex chromosomes in taxa experiencing recent speciation and diverging mainly via genetic drift. Here, we use nine sex‐linked and 21 autosomal genetic markers to investigate differential divergence and introgression between marker types in Certhia americana. We identified increased levels of genetic differentiation and reduced levels of gene flow on sex chromosomes relative to autosomes. This pattern is similar to those observed in other recently‐divergent avian species, providing another case study of the earlier role of sex chromosomes in divergence, relative to autosomes. Additionally, we identify three markers that may be under selection between Certhia americana lineages.     

Sex differences in structure and expression of the sex chromosome genes CHD1Z and CHD1W in zebra finches

Genes on the sex chromosomes are unique because of their sex-specific inheritance. One question is whether homologous gene pairs on the sex chromosomes, which have diverged in their sequence, have acquired different functions. We have analyzed the first homologous pair of genes (CHD1Z and CHD1W) discovered on the avian Z and W sex chromosomes of the zebra finch (Taeniopygia guttata) to examine whether functional differences may have evolved. Sequence analysis revealed that the two genes maintained a high degree of similarity especially within the C, H, and D domains, but outside of these regions larger differences were observed. Expression studies showed that CHD1W was unique to females and has the potential to produce a protein that CHD1Z does not. CHD1Z mRNA was expressed at a higher level in the male brain than in the female brain at various post-hatch ages. Reporter constructs containing the 5' flanking regions of each gene showed they had the ability to drive reporter expression in primary cell cultures. The 5' flanking region sequence of CHD1Z and CHD1W exhibited little homology, and differences in putative promoter elements were apparent. These differences between CHD1Z and CHD1W suggest that the two proteins may have diverged in their function.     

DMRT1 in a ratite bird: evidence for a role in sex determination and discovery of a putative regulatory element

Unlike mammals, birds have a ZZ male/ZW female sex-determining system. In most birds, the Z is large and gene rich, whereas the W is small and heterochromatic, but the ancient group of ratite birds are characterized by sex chromosomes that are virtually homomorphic. Any gene differentially present on the ratite Z and W is therefore a strong candidate for a sex-determining role. We have cloned part of the candidate bird sex-determining gene DMRT1 from the emu, a ratite bird, and have shown that it is expressed during the stages of development corresponding to gonadal differentiation in the chicken. The gene maps to the distal region of the Z short arm and is absent from the large W chromosome. Because most sequences on the emu W chromosome are shared with the Z, the Z-specific location constitutes strong evidence that differential dosage of DMRT1 is involved in sex determination in all birds. The sequence of emu DMRT1 has 88% homology with chicken DMRT1 and 65% with human DMRT1. Unexpectedly, an unexpressed 270-bp region in intron 3 of emu DMRT1 showed 90% homology with a sequence in the corresponding intron of human DMRT1. This extraordinarily high conservation across 300 million years of evolution suggests an important function, perhaps involved in control of DMRT1 expression and vertebrate sex determination.     

Different origins of bird and reptile sex chromosomes inferred from comparative mapping of chicken Z-linked genes

Recent progress of chicken genome projects has revealed that bird ZW and mammalian XY sex chromosomes were derived from different autosomal pairs of the common ancestor; however, the evolutionary relationship between bird and reptilian sex chromosomes is still unclear. The Chinese soft-shelled turtle (Pelodiscus sinensis) exhibits genetic sex determination, but no distinguishable (heteromorphic) sex chromosomes have been identified. In order to investigate this further, we performed molecular cytogenetic analyses of this species, and thereby identified ZZ/ZW-type micro-sex chromosomes. In addition, we cloned reptile homologues of chicken Z-linked genes from three reptilian species, the Chinese soft-shelled turtle and the Japanese four-striped rat snake (Elaphe quadrivirgata), which have heteromorphic sex chromosomes, and the Siam crocodile (Crocodylus siamensis), which exhibits temperature-dependent sex determination and lacks sex chromosomes. We then mapped them to chromosomes of each species using FISH. The linkage of the genes has been highly conserved in all species: the chicken Z chromosome corresponded to the turtle chromosome 6q, snake chromosome 2p and crocodile chromosome 3. The order of the genes was identical among the three species. The absence of homology between the bird Z chromosome and the snake and turtle Z sex chromosomes suggests that the origin of the sex chromosomes and the causative genes of sex determination are different between birds and reptiles.     

Secrets in the eyes of Black Oystercatchers: a new sexing technique

ABSTRACT.   Sexing oystercatchers in the field is difficult because males and females have identical plumage and are similar in size. Although Black Oystercatchers ( Haematopus bachmani ) are sexually dimorphic, using morphology to determine sex requires either capturing both pair members for comparison or using discriminant analyses to assign sex probabilistically based on morphometric traits. All adult Black Oystercatchers have bright yellow eyes, but some of them have dark specks, or eye flecks, in their irides. We hypothesized that this easily observable trait was sex-linked and could be used as a novel diagnostic tool for identifying sex. To test this, we compared data for oystercatchers from genetic molecular markers (CHD-W/CHD-Z and HINT-W/HINT-Z), morphometric analyses, and eye-fleck category (full eye flecks, slight eye flecks, and no eye flecks). Compared to molecular markers, we found that discriminant analyses based on morphological characteristics yielded variable results that were confounded by geographical differences in morphology. However, we found that eye flecks were sex-linked. Using an eye-fleck model where all females have full eye flecks and males have either slight eye flecks or no eye flecks, we correctly assigned the sex of 117 of 125 (94%) oystercatchers. Using discriminant analysis based on morphological characteristics, we correctly assigned the sex of 105 of 119 (88%) birds. Using the eye-fleck technique for sexing Black Oystercatchers may be preferable for some investigators because it is as accurate as discriminant analysis based on morphology and does not require capturing the birds.     

New primers for sex identification in the Chinese egret and other ardeid species

Using the universal P2/P8 primers, we were able to obtain the gene segments of chromo-helicase-DNA binding protein (CHD)-Z and CHD-W from ten species of ardeid birds including Chinese egret (Egretta eulophotes), little egret (E. garzetta), eastern reef egret (E. sacra), great egret (Ardea alba), grey heron (A. cinerea), Chinese pond-heron (Ardeola bacchus), cattle egret (Bubulcus ibis), black-crowned night-heron (Nycticorax nycticorax), cinnamon bittern (Ixobrychus cinnamomeus) and yellow bittern (I. sinensis). Based on conserved regions inside the P2/P8-derived sequences, we designed new PCR primers for sex identification in these ardeid species. Using agarose gel electrophoresis, the PCR products showed two bands for females (140 bp derived from CHD-W and the other 250 bp from CHD-ZW), whereas the males showed only the 250 bp band. The results indicated that our new primers could be used for accurate and convenient sex identification in ardeid species.     

Strong conservation of the bird Z chromosome in reptilian genomes is revealed by comparative painting despite 275 million years divergence

The divergence of lineages leading to extant squamate reptiles (lizards, snakes, and amphisbaenians) and birds occurred about 275 million years ago. Birds, unlike squamates, have karyotypes that are typified by the presence of a number of very small chromosomes. Hence, a number of chromosome rearrangements might be expected between bird and squamate genomes. We used chromosome-specific DNA from flow-sorted chicken (Gallus gallus) Z sex chromosomes as a probe in cross-species hybridization to metaphase spreads of 28 species from 17 families representing most main squamate lineages and single species of crocodiles and turtles. In all but one case, the Z chromosome was conserved intact despite very ancient divergence of sauropsid lineages. Furthermore, the probe painted an autosomal region in seven species from our sample with characterized sex chromosomes, and this provides evidence against an ancestral avian-like system of sex determination in Squamata. The avian Z chromosome synteny is, therefore, conserved albeit it is not a sex chromosome in these squamate species.     

A multi-gene approach reveals a complex evolutionary history in the Cyanistes species group

Quaternary climatic oscillations have been considered decisive in shaping much of the phylogeographic structure around the Mediterranean Basin. Within this paradigm, peripheral islands are usually considered as the endpoints of the colonization processes. Here, we use nuclear and mitochondrial markers to investigate the phylogeography of the blue tit complex (blue tit Cyanistes caeruleus, Canary blue tit C. teneriffae and azure tit C. cyanus), and assess the role of the Canary Islands for the geographic structuring of genetic variation. The Canary blue tit exhibits strong genetic differentiation within the Canary Islands and, in combination with other related continental species, provides an ideal model in which to examine recent differentiation within a closely related group of continental and oceanic island avian species. We analysed DNA sequences from 51 breeding populations and more than 400 individuals in the blue tit complex. Discrepancies in the nuclear and mitochondrial gene trees provided evidence of a complex evolutionary process around the Mediterranean Basin. Coalescent analyses revealed gene flow between C. caeruleus and C. teneriffae suggesting a dynamic process with multiple phases of colonization and geographic overlapping ranges. Microsatellite data indicated strong genetic differentiation among the Canary Islands and between the Canary archipelago and the close continental areas, indicating limited contemporary gene flow. Diversification of the blue tit complex is estimated to have started during the early Pliocene (≈ 5 Ma), coincident with the end of Messinian salinity crisis. Phylogenetic analyses indicated that the North African blue tit is derived from the Canary blue tits, a pattern is avian 'back colonization' that contrasts with more traditionally held views of islands being sinks rather than sources.     

No Evidence for a Second Evolutionary Stratum during the Early Evolution of Mammalian Sex Chromosomes

Mammalian sex chromosomes originated from a pair of autosomes, and homologous genes on the sex chromosomes (gametologs) differentiated through recombination arrest between the chromosomes. It was hypothesized that this differentiation in eutherians took place in a stepwise fashion and left a footprint on the X chromosome termed “evolutionary strata.” The evolutionary stratum hypothesis claims that strata 1 and 2 (which correspond to the first two steps of chromosomal differentiation) were generated in the stem lineage of Theria or before the divergence between eutherians and marsupials. However, this prediction relied solely on the molecular clock hypothesis between pairs of human gametologs, and molecular evolution of marsupial sex chromosomal genes has not yet been investigated. In this study, we analyzed the following 7 pairs of marsupial gametologs, together with their eutherian orthologs that reside in stratum 1 or 2: SOX3/SRY, RBMX/Y, RPS4X/Y, HSFX/Y, XKRX/Y, SMCX/Y (KDM5C/D, JARID1C/D), and UBE1X/Y (UBA1/UBA1Y). Phylogenetic analyses and estimated divergence time of these gametologs reveal that they all differentiated at the same time in the therian ancestor. We have also provided strong evidence for gene conversion that occurred in the 3′ region of the eutherian stratum 2 genes (SMCX/Y and UBE1X/Y). The results of the present study show that (1) there is no compelling evidence for the second stratum in the stem lineage of Theria; (2) gene conversion, which may have occurred between SMCX/Y and UBE1X/Y in the eutherian lineage, potentially accounts for their apparently lower degree of overall divergence.