The Staurotypus Turtles and Aves Share the Same Origin of Sex Chromosomes but Evolved Different Types of Heterogametic Sex Determination

Reptiles have a wide diversity of sex-determining mechanisms and types of sex chromosomes. Turtles exhibit temperature-dependent sex determination and genotypic sex determination, with male heterogametic (XX/XY) and female heterogametic (ZZ/ZW) sex chromosomes. Identification of sex chromosomes in many turtle species and their comparative genomic analysis are of great significance to understand the evolutionary processes of sex determination and sex chromosome differentiation in Testudines. The Mexican giant musk turtle (Staurotypus triporcatus, Kinosternidae, Testudines) and the giant musk turtle (Staurotypus salvinii) have heteromorphic XY sex chromosomes with a low degree of morphological differentiation; however, their origin and linkage group are still unknown. Cross-species chromosome painting with chromosome-specific DNA from Chinese soft-shelled turtle (Pelodiscus sinensis) revealed that the X and Y chromosomes of S. triporcatus have homology with P. sinensis chromosome 6, which corresponds to the chicken Z chromosome. We cloned cDNA fragments of S. triporcatus homologs of 16 chicken Z-linked genes and mapped them to S. triporcatus and S. salvinii chromosomes using fluorescence in situ hybridization. Sixteen genes were localized to the X and Y long arms in the same order in both species. The orders were also almost the same as those of the ostrich (Struthio camelus) Z chromosome, which retains the primitive state of the avian ancestral Z chromosome. These results strongly suggest that the X and Y chromosomes of Staurotypus turtles are at a very early stage of sex chromosome differentiation, and that these chromosomes and the avian ZW chromosomes share the same origin. Nonetheless, the turtles and birds acquired different systems of heterogametic sex determination during their evolution.     

Inference of the Protokaryotypes of Amniotes and Tetrapods and the Evolutionary Processes of Microchromosomes from Comparative Gene Mapping

Comparative genome analysis of non-avian reptiles and amphibians provides important clues about the process of genome evolution in tetrapods. However, there is still only limited information available on the genome structures of these organisms. Consequently, the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes in tetrapods remain poorly understood. We constructed chromosome maps of functional genes for the Chinese soft-shelled turtle (Pelodiscus sinensis), the Siamese crocodile (Crocodylus siamensis), and the Western clawed frog (Xenopus tropicalis) and compared them with genome and/or chromosome maps of other tetrapod species (salamander, lizard, snake, chicken, and human). This is the first report on the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes inferred from comparative genomic analysis of vertebrates, which cover all major non-avian reptilian taxa (Squamata, Crocodilia, Testudines). The eight largest macrochromosomes of the turtle and chicken were equivalent, and 11 linkage groups had also remained intact in the crocodile. Linkage groups of the chicken macrochromosomes were also highly conserved in X. tropicalis, two squamates, and the salamander, but not in human. Chicken microchromosomal linkages were conserved in the squamates, which have fewer microchromosomes than chicken, and also in Xenopus and the salamander, which both lack microchromosomes; in the latter, the chicken microchromosomal segments have been integrated into macrochromosomes. Our present findings open up the possibility that the ancestral amniotes and tetrapods had at least 10 large genetic linkage groups and many microchromosomes, which corresponded to the chicken macro- and microchromosomes, respectively. The turtle and chicken might retain the microchromosomes of the amniote protokaryotype almost intact. The decrease in number and/or disappearance of microchromosomes by repeated chromosomal fusions probably occurred independently in the amphibian, squamate, crocodilian, and mammalian lineages.     

Long-term sex reversal by oestradiol in amniotes with heteromorphic sex chromosomes

Oestradiol application during embryonic development reverses the sex of male embryos and results in normal female differentiation in reptiles lacking heteromorphic sex chromosomes, but fails to do so in birds and mammals with heteromorphic sex chromosomes. It is not clear whether the evolution of heteromorphic sex chromosomes in amniotes is accompanied by insensitivity to oestradiol, or if the association between oestradiol insensitivity and heteromorphic sex chromosomes can be attributable to phylogenetic constraints in these taxa. Turtles provide an ideal system to examine the potential relationship between oestradiol insensitivity and sex chromosome heteromorphy, since there are species with heteromorphic sex chromosomes that are closely related to species lacking heteromorphic sex chromosomes. We investigated this relationship by examining the long-term effects of oestradiol-17beta application on sex determination in Staurotypus triporcatus and Staurotypus salvinii, two turtle species with male heterogamety. After raising the turtles in the lab for 3 years, we found follicular and Müllerian duct morphology in oestradiol-treated turtles that was identical to that of untreated females. The lasting sex reversal suggests that the evolutionary transition between systems lacking heteromorphic sex chromosomes and those with heteromorphic sex chromosomes is not constrained by a fundamental mechanistic difference.     

Chromosome banding pattern conservatism in birds and nonhomology of chromosome banding patterns between birds,turtles, snakes and amphibians

The G-banded karyotypes of 4 species of birds representing the orders Galliformes, Columbiformes and Musophagiformes were compared. Banding pattern homology between orders was limited t 5o 5 major chromosome arms and the Z chromosome. Even in these major chromosome arms pericentric and paracentric inversions produced alteration of the banding pattern sequences. Addition of constitutive heterochromatin was responsible for changes in banding patterns in the Z chromosome. The chromosome banding patterns of an emydid turtle, Terrepene carolina, 5 species of boid snakes of the genera Liasis, Acrantophis, and Sanzinia and the African clawed-frog. Xenopus muelleri, were also compared to the bird chromosome banding patterns. No homology was observed between any of these major groups: bird, snake, turtle, amphibian. However, intergroup homology was apparent. - The data obtained do not support reports of broad interordinal direct homology of the macrochromosomes of birds and refutes the idea of a primitive bird karyotype with 3 pairs of "Agroup' chromosomes and 3 pairs of "B group' chromosomes. - The major mechanisms responsible for chromosome evolution in birds appear to be centric and tandem fusions, paracentric and pericentric inversions, and addition or deletion of heterochromatin.     

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.     

The origin and differentiation of the heteromorphic sex chromosomes Z, W, X, and Y in the frog Rana rugosa, inferred from the sequences of a sex-linked gene, ADP/ATP translocase

Sex chromosomes of the Japanese frog Rana rugosa are heteromorphic in the male (XX/XY) or in the female (ZZ/ZW) in two geographic forms, whereas they are still homomorphic in both sexes in two other forms (Hiroshima and Isehara types). To make clear the origin and differentiation mechanisms of the heteromorphic sex chromosomes, we isolated a sex-linked gene, ADP/ATP translocase, and constructed a phylogenetic tree of the genes derived from the sex chromosomes. The tree shows that the Hiroshima gene diverges first, and the rest form two clusters: one includes the Y and Z genes and the other includes the X, W, and Isehara genes. The Hiroshima gene shares more sequence similarity with the Y and Z genes than with the X, W, and Isehara genes. This suggests that the Y and Z sex chromosomes originate from the Hiroshima type, whereas the X and W chromosomes originate from the Isehara-type sex chromosome. Thus, we infer that hybridization between two ancestral forms, with the Hiroshima-type sex chromosome in one and the Isehara-type sex chromosome in the other, was the primary event causing differentiation of the heteromorphic sex chromosomes.      

Isolation and characterisation of crocodile and python ovotransferrins

Transferrins play a major role in iron homeostasis and metabolism. In vertebrates, these proteins are synthesised in the liver and dispersed within the organism by the bloodstream. In oviparous vertebrates additional expression is observed in the oviduct and the synthesised protein is deposited in egg white as ovotransferrin. Most research on ovotransferrin has been performed on the chicken protein. There is a limited amount of information on other bird transferrins, and until our previous paper on red-eared turtle protein there was no data on the isolation, sequencing and biochemical properties of reptilian ovotransferrins. Recently our laboratory deposited ten new sequences of reptilian transferrins in the EMBL database. A comparative analysis of these sequences indicates a possibility of different mechanisms of iron release among crocodile and snake transferrin. In the present paper we follow with the purification and analysis of the basic biochemical properties of two crocodile (Crocodilus niloticus, C. rhombifer) and one snake (Python molurus bivittatus) ovotransferrins. The proteins were purified by anion exchange and hydrophobic chromatography, and their N-terminal amino-acid sequences, molecular mass and isoelectric points were determined. All three proteins are glycosylated and their N-glycan chromatographic profiles show the largest contribution of neutral oligosaccharides in crocodile and disialylated glycans in python ovotransferrin. The absorption spectra of iron-saturated transferrins were analysed. Iron release from these proteins is pH-dependent, showing a biphasic character in crocodile ovotransferrins and a monophasic type in the python protein. The reason for the different types of iron release is discussed.     

Cytogenetics of the Javan file snake (Acrochordus javanicus) and the evolution of snake sex chromosomes

Advanced snakes (Caenophidia) are an important group including around 90% of the recent species of snakes. The basal splitting of the clade is still rather controversial, and it is not fully understood when the differentiation of sex chromosomes started in snake evolution. To help resolve these questions, we performed cytogenetic analysis on the Javan file snake, also known as the elephant trunk snake (Acrochordus javanicus) from the family Acrochordidae, which occupies an informative phylogenetic position. For the first time for acrochordids, we identified heteromorphic ZZ/ZW sex chromosomes with a highly heterochromatic W chromosome. These traits are likely synapomorphies of advanced snakes. In contrast to other caenophidian snakes, the Javan file snake lacks an accumulation of Bkm repeats and interstitial telomeric repeats on the W chromosome. This observation supports the sister group relationship between acrochordids and all other caenophidian snakes including the family Xenodermatidae and questions the suggested role of Bkm repeats in the formation of sex heterochromatin in snakes. The revealed partial gene content of the Z chromosome in acrochordids supports the hypothesis that the progressive degeneration of the W chromosome commenced in snakes before the basal split of Caenophidia, albeit its evolutionary rate in file snakes might be slower than in their sister lineage.     

A new look at the evolution of avian sex chromosomes

Birds have a ubiquitous, female heterogametic, ZW sex chromosome system. The current model suggests that the Z chromosome and its degraded partner, the W chromosome, evolved from an ancestral pair of autosomes independently from the mammalian XY male heteromorphic sex chromosomes--which are similar in size, but not gene content (Graves, 1995; Fridolfsson et al., 1998). Furthermore the degradation of the W has been proposed to be progressive, with the basal clade of birds (the ratites) possessing virtually homomorphic sex chromosomes and the more recently derived birds (the carinates) possessing highly heteromorphic sex chromosomes (Ohno, 1967; Solari, 1993). Recent findings have suggested an alternative to independent evolution of bird and mammal chromosomes, in which an XY system took over directly from an ancestral ZW system. Here we examine recent research into avian sex chromosomes and offer alternative suggestions as to their evolution.     

Genetic mapping in a natural population of collared flycatchers (Ficedula albicollis): conserved synteny but gene order rearrangements on the avian Z chromosome

Data from completely sequenced genomes are likely to open the way for novel studies of the genetics of nonmodel organisms, in particular when it comes to the identification and analysis of genes responsible for traits that are under selection in natural populations. Here we use the draft sequence of the chicken genome as a starting point for linkage mapping in a wild bird species, the collared flycatcher - one of the most well-studied avian species in ecological and evolutionary research. A pedigree of 365 flycatchers was established and genotyped for single nucleotide polymorphisms in 23 genes selected from (and spread over most of) the chicken Z chromosome. All genes were also found to be located on the Z chromosome in the collared flycatcher, confirming conserved synteny at the level of gene content across distantly related avian lineages. This high degree of conservation mimics the situation seen for the mammalian X chromosome and may thus be a general feature in sex chromosome evolution, irrespective of whether there is male or female heterogamety. Alternatively, such unprecedented chromosomal conservation may be characteristic of most chromosomes in avian genome evolution. However, several internal rearrangements were observed, meaning that the transfer of map information from chicken to nonmodel bird species cannot always assume conserved gene orders. Interestingly, the rate of recombination on the Z chromosome of collared flycatchers was only approximately 50% that of chicken, challenging the widely held view that birds generally have high recombination rates.     

Heteromorphic Z and W sex chromosomes in Physalaemus ephippifer (Steindachner, 1864) (Anura, Leiuperidae)

Heteromorphisms between sex chromosomes are rarely found in anurans and sex chromosome differentiation is considered to be a set of recent recurrent events in the evolutionary history of this group. This paper describes for the first time heteromorphic sex chromosomes Z and W in the leiuperid genus Physalaemus. They were found in P. ephippifer, a species of the P. cuvieri group, and corresponded to the eighth pair of its karyotype. The W chromosome differed from the Z chromosome by the presence of an additional segment in the short arm, composed of a distal NOR and an adjacent terminal DAPI-positive C-band. The identification of this sex chromosome pair may help in future investigations into the sex determining genes in the genus Physalaemus.     

ANOLIS SEX CHROMOSOMES ARE DERIVED FROM A SINGLE ANCESTRAL PAIR

To explain the frequency and distribution of heteromorphic sex chromosomes in the lizard genus Anolis, we compared the relative roles of sex chromosome conservation versus turnover of sex‐determining mechanisms. We used model‐based comparative methods to reconstruct karyotype evolution and the presence of heteromorphic sex chromosomes onto a newly generated Anolis phylogeny. We found that heteromorphic sex chromosomes evolved multiple times in the genus. Fluorescent in situ hybridization (FISH) of repetitive DNA showed variable rates of Y chromosome degeneration among Anolis species and identified previously undetected, homomorphic sex chromosomes in two species. We confirmed homology of sex chromosomes in the genus by performing FISH of an X‐linked bacterial artificial chromosome (BAC) and quantitative PCR of X‐linked genes in multiple Anolis species sampled across the phylogeny. Taken together, these results are consistent with long‐term conservation of sex chromosomes in the group. Our results pave the way to address additional questions related to Anolis sex chromosome evolution and describe a conceptual framework that can be used to evaluate the origins and evolution of heteromorphic sex chromosomes in other clades.     

Cytogenetic analysis of California condor (Gymnogyps californianus) chromosomes: comparison with chicken (Gallus gallus) macrochromosomes

The California condor is the largest flying bird in North America and belongs to a group of New World vultures. Recovering from a near fatal population decline, and currently with only 197 extant individuals, the species remains listed as endangered. Very little genetic information exists for this species, although sexing methods employing chromosome analysis or W-chromosome specific amplification is routinely applied for the management of this monomorphic species. Keeping in mind that genetic conditions like chondrodystrophy have been identified, preliminary steps were undertaken in this study to understand the genome organization of the condor. This included an extensive cytogenetic analysis that provided (i) a chromosome number of 80 (with a likelihood of an extra pair of microchromosomes), and (ii) information on the centromeres, telomeres and nucleolus organizer regions. Further, a comparison between condor and chicken macrochromosomes was obtained by using individual chicken chromosome specific paints 1-9 and Z and W on condor metaphase spreads. Except for chromosomes 4 and Z, each of the chicken (GGA) macrochromosomes painted a single condor (GCA) macrochromosome. GGA4 paint detected complete homology with two condor chromosomes, viz., GCA4 and GCA9 providing additional proof that the latter are ancestral chromosomes in the birds. The chicken Z chromosome showed correspondence with both Z and W in the condor. The homology suggests that the condor sex chromosomes have not completely differentiated during evolution, which is unlike the majority of the non-ratites studied up till now. Overall, the study provides detailed cytogenetic and basic comparative information on condor chromosomes. These findings significantly advance the effort to study the chondrodystrophy that is responsible for over ten percent mortality in the condor.     

Independent Evolution of Transcriptional Inactivation on Sex Chromosomes in Birds and Mammals

X chromosome inactivation in eutherian mammals has been thought to be tightly controlled, as expected from a mechanism that compensates for the different dosage of X-borne genes in XX females and XY males. However, many X genes escape inactivation in humans, inactivation of the X in marsupials is partial, and the unrelated sex chromosomes of monotreme mammals have incomplete and gene-specific inactivation of X-linked genes. The bird ZW sex chromosome system represents a third independently evolved amniote sex chromosome system with dosage compensation, albeit partial and gene-specific, via an unknown mechanism (i.e. upregulation of the single Z in females, down regulation of one or both Zs in males, or a combination). We used RNA-fluorescent in situ hybridization (RNA-FISH) to demonstrate, on individual fibroblast cells, inactivation of 11 genes on the chicken Z and 28 genes on the X chromosomes of platypus. Each gene displayed a reproducible frequency of 1Z/1X-active and 2Z/2X-active cells in the homogametic sex. Our results indicate that the probability of inactivation is controlled on a gene-by-gene basis (or small domains) on the chicken Z and platypus X chromosomes. This regulatory mechanism must have been exapted independently to the non-homologous sex chromosomes in birds and mammals in response to an over-expressed Z or X in the homogametic sex, highlighting the universal importance that (at least partial) silencing plays in the evolution on amniote dosage compensation and, therefore, the differentiation of sex chromosomes.     

Dynamic sex chromosomes in Old World chameleons (Squamata: Chamaeleonidae)

Much of our current state of knowledge concerning sex chromosome evolution is based on a handful of ‘exceptional’ taxa with heteromorphic sex chromosomes. However, classifying the sex chromosome systems of additional species lacking easily identifiable, heteromorphic sex chromosomes is indispensable if we wish to fully understand the genesis, degeneration and turnover of vertebrate sex chromosomes. Squamate reptiles (lizards and snakes) are a potential model clade for studying sex chromosome evolution as they exhibit a suite of sex‐determining modes yet most species lack heteromorphic sex chromosomes. Only three (of 203) chameleon species have identified sex chromosome systems (all with female heterogamety, ZZ/ZW). This study uses a recently developed method to identify sex‐specific genetic markers from restriction site‐associated DNA sequence (RADseq) data, which enables the identification of sex chromosome systems in species lacking heteromorphic sex chromosomes. We used RADseq and subsequent PCR validation to identify an XX/XY sex chromosome system in the veiled chameleon (Chamaeleo calyptratus), revealing a novel transition in sex chromosome systems within the Chamaeleonidae. The sex‐specific genetic markers identified here will be essential in research focused on sex‐specific, comparative, functional and developmental evolutionary questions, further promoting C. calyptratus’ utility as an emerging model organism.     

Faster evolution of Z-linked duplicate genes in chicken

It has been shown that duplicate genes on the X chromosome evolve much faster than duplicate genes on autosomes in Drosophila melanogaster.However,whether this phenomenon is general and can be applied to other species is not known.Here we examined this issue in chicken that have heterogametic females(females have ZW sex chromosome).We compared sequence divergence of duplicate genes on the Z chromosome with those on autosomes.We found that duplications on the Z chromosome indeed evolved faster than those on autosomes and show distinct patterns of molecular evolution from autosomal duplications.Examination of the expression of duplicate genes revealed an enrichment of duplications on the Z chromosome having male-biased expression and an enrichment of duplications on the autosomes having female-biased expression.These results suggest an evolutionary trend of the recruitment of duplicate genes towards reproduction-specific function.The faster evolution of duplications on Z than on the autosomes is most likely contributed by the selective forces driving the fixation of adaptive mutations on Z.Therefore,the common phenomena observed in both flies and chicken suggest that duplicate genes on sex chromosomes have distinct dynamics and are more influenced by natural selection than antosomal duplications,regardless of the kind of sex determination systems.