Denis O'Meally

Sex chromosome evolution in lizards: independent origins and rapid transitions.

Reptiles epitomize the variability of reproductive and sex determining modes and mechanisms among amniotes. These modes include gonochorism (separate sexes) and parthenogenesis, oviparity, viviparity, and ovoviviparity, genotypic sex determination (GSD) with male (XX/XY) and female (ZZ/ZW) heterogamety and temperature-dependent sex determination (TSD). Lizards (order Squamata, suborder Sauria) are particularly fascinating because the distribution of sex-determining mechanisms shows no clear phylogenetic segregation. This implies that there have been multiple transitions between TSD and GSD, and between XY and ZW sex chromosome systems. Approximately 1,000 species of lizards have been karyotyped and among those, fewer than 200 species have sex chromosomes, yet they display remarkable diversity in morphology and degree of degeneration. The high diversity of sex chromosomes as well as the presence of species with TSD, imply multiple and independent origins of sex chromosomes, and suggest that the mechanisms of sex determination are extremely labile in lizards. In this paper, we review the current state of knowledge of sex chromosomes in lizards and the distribution of sex determining mechanisms and sex chromosome forms within and among families. We establish for the first time an association between the occurrence of female heterogamety and TSD within lizard families, and propose mechanisms by which female heterogamety and TSD may have co-evolved. We suggest that lizard sex determination may be much more the result of an interplay between sex chromosomes and temperature than previously thought, such that the sex determination mode is influenced by the nature of heterogamety as well as temperature sensitivity and the stage of sex chromosome degeneration.

Sex reversal triggers the rapid transition from genetic to temperature-dependent sex

Sex determination in animals is amazingly plastic. Vertebrates display contrasting strategies ranging from complete genetic control of sex (genotypic sex determination) to environmentally determined sex (for example, temperature-dependent sex determination). Phylogenetic analyses suggest frequent evolutionary transitions between genotypic and temperature-dependent sex determination in environmentally sensitive lineages, including reptiles. These transitions are thought to involve a genotypic system becoming sensitive to temperature, with sex determined by gene–environment interactions. Most mechanistic models of transitions invoke a role for sex reversal. Sex reversal has not yet been demonstrated in nature for any amniote, although it occurs in fish and rarely in amphibians. Here we make the first report of reptile sex reversal in the wild, in the Australian bearded dragon (Pogona vitticeps), and use sex-reversed animals to experimentally induce a rapid transition from genotypic to temperature-dependent sex determination. Controlled mating of normal males to sex-reversed females produces viable and fertile offspring whose phenotypic sex is determined solely by temperature (temperature-dependent sex determination). The W sex chromosome is eliminated from this lineage in the first generation. The instantaneous creation of a lineage of ZZ temperature-sensitive animals reveals a novel, climate-induced pathway for the rapid transition between genetic and temperature-dependent sex determination, and adds to concern about adaptation to rapid global climate change.

Non-homologous sex chromosomes of birds and snakes share repetitive sequences

Snake sex chromosomes provided Susumo Ohno with the material on which he based his theory of how sex chromosomes differentiate from autosomal pairs. Like birds, snakes have a ZZ male/ZW female sex chromosome system, in which the snake Z is a macrochromosome much the same size as the bird Z. However, the gene content shows clearly that the snake and bird Z chromosomes are completely non-homologous. The molecular aspect of W chromosome degeneration in snakes remains largely unexplored. We used comparative genomic hybridization to identify the female-specific region of the W chromosome in representative species of Australian snakes. Using this approach, we show that an increasingly complex suite of repeats accompanies the evolution of W chromosome heteromorphy. In particular, we found that while the python Liasis fuscus exhibits no sex-specific repeats and indeed, no cytologically recognizable sex-specific region, the colubrid Stegonotus cucullatus shows a large domain on the short arm of the W chromosome that consists of female-specific repeats, and the large W of Notechis scutatus is composed almost entirely of repetitive sequences, including Bkm and 18S rDNA-related elements. FISH mapping of both simple and complex probes shows patterns of repeat amplification concordant with the size of the female-specific region in each species examined. Mapping of intronic sequences of genes that are sex-linked in both birds (DMRT1) and snakes (CTNNB1) reveals massive amplification in discrete domains on the W chromosome of the elapid N. scutatus. Using chicken W chromosome paint, we demonstrate that repetitive sequences are shared between the sex chromosomes of birds and derived snakes. This could be explained by ancestral but as yet undetected shared synteny of bird and snake sex chromosomes or may indicate functional homology of the repeats and suggests that degeneration is a convergent property of sex chromosome evolution. We also establish that synteny of snake Z-linked genes has been conserved for at least 166 million years and that the snake Z consists of two conserved blocks derived from the same ancestral vertebrate chromosome.

Are some chromosomes particularly good at sex? Insights from amniotes

Several recent studies have produced comparative maps of genes on amniote sex chromosomes, revealing homology of gene content and arrangement across lineages as divergent as mammals and lizards. For example, the chicken Z chromosome, which shares homology with the sex chromosomes of all birds, monotremes, and a gecko, is a striking example of stability of genome organization and retention, or independent acquisition, of function in sex determination. In other lineages, such as snakes and therian mammals, well conserved but independently evolved sex chromosome systems have arisen. Among lizards, novel sex chromosomes appear frequently, even in congeneric species. Here, we review recent gene mapping data, examine the evolutionary relationships of amniote sex chromosomes and argue that gene content can predispose some chromosomes to a specialized role in sex determination.

Evolutionary history of novel genes on the tammar wallaby Y chromosome: Implications for sex chromosome evolution

We report here the isolation and sequencing of 10 Y-specific tammar wallaby (Macropus eugenii) BAC clones, revealing five hitherto undescribed tammar wallaby Y genes (in addition to the five genes already described) and several pseudogenes. Some genes on the wallaby Y display testis-specific expression, but most have low widespread expression. All have partners on the tammar X, along with homologs on the human X. Nonsynonymous and synonymous substitution ratios for nine of the tammar XY gene pairs indicate that they are each under purifying selection. All 10 were also identified as being on the Y in Tasmanian devil (Sarcophilus harrisii; a distantly related Australian marsupial); however, seven have been lost from the human Y. Maximum likelihood phylogenetic analyses of the wallaby YX genes, with respective homologs from other vertebrate representatives, revealed that three marsupial Y genes (HCFC1X/Y, MECP2X/Y, and HUWE1X/Y) were members of the ancestral therian pseudoautosomal region (PAR) at the time of the marsupial/eutherian split; three XY pairs (SOX3/SRY, RBMX/Y, and ATRX/Y) were isolated from each other before the marsupial/eutherian split, and the remaining three (RPL10X/Y, PHF6X/Y, and UBA1/UBE1Y) have a more complex evolutionary history. Thus, the small marsupial Y chromosome is surprisingly rich in ancient genes that are retained in at least Australian marsupials and evolved from testis-brain expressed genes on the X.

Amplification of microsatellite repeat motifs is associated with the evolutionary differentiation and heterochromatinization of sex chromosomes in Sauropsida

The sex chromosomes in Sauropsida (reptiles and birds) have evolved independently many times. They show astonishing diversity in morphology ranging from cryptic to highly differentiated sex chromosomes with male (XX/XY) and female heterogamety (ZZ/ZW). Comparing such diverse sex chromosome systems thus provides unparalleled opportunities to capture evolution of morphologically differentiated sex chromosomes in action. Here, we describe chromosomal mapping of 18 microsatellite repeat motifs in eight species of Sauropsida. More than two microsatellite repeat motifs were amplified on the sex-specific chromosome, W or Y, in five species (Bassiana duperreyi, Aprasia parapulchella, Notechis scutatus, Chelodina longicollis, and Gallus gallus) of which the sex-specific chromosomes were heteromorphic and heterochromatic. Motifs (AAGG)n and (ATCC)n were amplified on the W chromosome of Pogona vitticeps and the Y chromosome of Emydura macquarii, respectively. By contrast, no motifs were amplified on the W chromosome of Christinus marmoratus, which is not much differentiated from the Z chromosome. Taken together with previously published studies, our results suggest that the amplification of microsatellite repeats is tightly associated with the differentiation and heterochromatinization of sex-specific chromosomes in sauropsids as well as in other taxa. Although some motifs were common between the sex-specific chromosomes of multiple species, no correlation was observed between this commonality and the species phylogeny. Furthermore, comparative analysis of sex chromosome homology and chromosomal distribution of microsatellite repeats between two closely related chelid turtles, C. longicollis and E. macquarii, identified different ancestry and differentiation history. These suggest multiple evolutions of sex chromosomes in the Sauropsida.

The First Cytogenetic Map of the Tuatara, Sphenodon punctatus

Tuatara, Sphenodon punctatus, is the last survivor of the distinctive reptilian order Rhynchocephalia and is a species of extraordinary zoological interest, yet only recently have genomic analyses been undertaken. The karyotype consists of 28 macrochromosomes and 8 microchromosomes. A Bacterial Artificial Chromosome (BAC) library constructed for this species has allowed the first characterization of the tuatara genome. Sequence analysis of 11 fully sequenced BAC clones (∼0.03% coverage) increased the estimate of genome wide GC composition to 47.8%, the highest reported for any vertebrate. Our physical mapping data demonstrate discrete accumulation of repetitive elements in large blocks on some chromosomes, particularly the microchromosomes. We suggest that the large size of the genome (5.0 pg/haploid) is due to the accumulation of repetitive sequences. The microchromosomes of tuatara are rich in repetitive sequences, and the observation of one animal that lacked a microchromosome pair suggests that at least this microchromosome is unnecessary for survival. We used BACs bearing orthologues of known genes to construct a low-coverage cytogenetic map containing 21 markers. We identified a region on chromosome 4 of tuatara that shares homology with 7 Mb of chicken chromosome 2, and therefore the orthologous region of the snake Z chromosome. We identified a region on tuatara chromosome 3 that is orthologous to the chicken Z, and a region on chromosome 9 orthologous to the mammalian X. Since the tuatara determines sex by temperature and has no sex chromosomes, this implies that different tuatara autosome regions are homologous with the sex chromosomes of mammals, birds and snakes. We have identified anchor BAC clones that can be used to reliably mark chromosomes 3–7, 10 and 13, some of which are difficult to distinguish based on morphology alone. Fluorescence in situ hybridization mapping of 18S rDNA confirms the presence of a single NOR located on the long arm of chromosome 7, as previously identified by silver staining. Further work to construct a dense physical map will lead to a better understanding of the dynamics of genome evolution and organization in this isolated species.

Sex Reversal in Reptiles: Reproductive Oddity or Powerful Driver of Evolutionary Change?

Is sex a product of genes, the environment, or both? In this review, we describe the diversity of sex-determining mechanisms in reptiles, with a focus on systems that display gene-environment interactions. We summarise the field and laboratory-based evidence for the occurrence of environmental sex reversal in reptiles and ask whether this is a widespread evolutionary mechanism affecting the evolution of sex chromosomes and speciation in vertebrates. Sex determination systems exist across a continuum of genetic and environmental influences, blurring the lines between what was once considered a strict dichotomy between genetic sex determination and temperature-dependent sex determination. Across this spectrum, we identify the potential for sex reversal in species with clearly differentiated heteromorphic sex chromosomes (Pogona vitticeps, Bassiana duperreyi, Eremias multiocellata, Gekko japonicus), weakly differentiated homomorphic sex chromosomes (Niveoscincus ocellatus), and species with only a weak heritable predisposition for sex (Emys orbicularis, Trachemys scripta). We argue that sex reversal is widespread in reptiles (Testudines, Lacertidae, Agamidae, Scincidae, Gekkonidae) and has the potential to have an impact on individual fitness, resulting in reproductively, morphologically, and behaviourally unique phenotypes. Sex reversal is likely to be a powerful evolutionary force responsible for generating and maintaining lability and diversity in reptile sex-determining modes.

Molecular evolution of Dmrt1 accompanies change of sex-determining mechanisms in reptilia.

In reptiles, sex-determining mechanisms have evolved repeatedly and reversibly between genotypic and temperature-dependent sex determination. The gene Dmrt1 directs male determination in chicken (and presumably other birds), and regulates sex differentiation in animals as distantly related as fruit flies, nematodes and humans. Here, we show a consistent molecular difference in Dmrt1 between reptiles with genotypic and temperature-dependent sex determination. Among 34 non-avian reptiles, a convergently evolved pair of amino acids encoded by sequence within exon 2 near the DM-binding domain of Dmrt1 distinguishes species with either type of sex determination. We suggest that this amino acid shift accompanied the evolution of genotypic sex determination from an ancestral condition of temperature-dependent sex determination at least three times among reptiles, as evident in turtles, birds and squamates. This novel hypothesis describes the evolution of sex-determining mechanisms as turnover events accompanied by one or two small mutations.

Contents Vol. 10, 2016

Karin Schmid (address as for M. Schmid) E-mail: karin.schmid@uni-wuerzburg.de Peter Koopman Professor of Developmental Biology Institute for Molecular Bioscience The University of Queensland AU–Brisbane, Qld. 4072 (Australia) Tel. (+61) 7 3346 2059; Fax. (+61) 7 3346 2101 E-mail p.koopman@imb.uq.edu.au Manfred Schartl Institute of Physiological Chemistry I University of Würzburg Biozentrum, Am Hubland D–97074 Würzburg (Germany) Tel. (+49) 931 318 4148; Fax (+49) 931 318 4150 E-mail: phch1@biozentrum.uni-wuerzburg.de