<p>The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly <italic>in vivo</italic>. The gonad is an ideal place to study this process because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. The early gonad is composed of four lineages: supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. Each lineage in the early gonad consists of bipotential progenitors capable of adopting either a male or female fate, which they do in a coordinated manner to form a functional testis or ovary. The supporting cell lineage is of particular interest because the decision of these cells to adopt the male or female fate dictates the fate of the gonad as a whole. </p><p><p>To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells of the four lineages from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. Our analysis identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We also determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. Multipotent progenitors that show lineage priming express markers of the various fates into which they can differentiate and subsequently silence genes associated with the fate not adopted as they differentiate. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cell progenitors were primed with a female bias. This yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates. </p><p><p>We also used a genetic approach to investigate how individual factors contribute to the adoption of the male supporting cell fate. We previously demonstrated that <italic>Fgf9</italic> and <italic>Wnt4</italic> act as mutually antagonistic factors to promote male or female development of the bipotential mammalian gonad. <italic>Fgf9</italic> is necessary to maintain <italic>Sox9</italic> expression, which drives male development. However, whether FGF9 acted directly on <italic>Sox9</italic> or indirectly through repression of <italic>Wnt4</italic>, was unknown. <italic>Wnt4</italic> is a female-primed gene, and is therefore repressed during male development. To determine how <italic>Fgf9</italic> functioned, we generated double <italic>Fgf9/Wnt4</italic> and <italic>Fgfr2/Wnt4</italic> mutants. While single XY <italic>Fgf9</italic> and <italic>Fgfr2</italic> mutants showed partial or complete male-to-female sex reversal, loss of <italic>Wnt4</italic> in an <italic>Fgf9</italic> or <italic>Fgfr2</italic> mutant background rescued normal testis development. We also found that <italic>Wnt4</italic> and another female-associated gene (<italic>Rspo1</italic>) were derepressed in <italic>Fgf9</italic> mutants prior to the down-regulation of <italic>Sox9</italic>. Thus, the primary function of <italic>Fgf9</italic> is the repression of female genes, including <italic>Wnt4</italic>. We also tested the reciprocal possibility: that de-repression of <italic>Fgf9</italic> was responsible for the aspects of male development observed in XX <italic>Wnt4</italic> mutants. However, we show that loss of <italic>Fgf9</italic> in XX <italic>Wnt4<super>-/-</super></italic> gonads does not rescue the partial female-to-male sex reversal. </p><p><p>Based on the <italic>Fgf9/Wnt4</italic> double mutant studies, we propose a two part model of male sex determination in which both the activation of male genes and repression of female genes is required. Also, this work demonstrates that the repression of the female-primed gene <italic>Wnt4</italic> is required for male development, and <italic>Fgf9</italic> is one factor that leads to the repression of female-primed genes.</p> / Dissertation

<p>Sex determination is a critical biological process for all sexually reproducing animals. Despite its significance, evolution has provided a vast array of mechanisms by which sexual phenotype is determined and elaborated even within amniote vertebrates. The most prevalent systems of sex determination in this clade are genetic and temperature dependent sex determination. These two systems are sometimes consistent within large groups of species, such as the mammals who nearly ubiquitously utilize XY genetic sex determination, or they can be much more mixed as in reptiles that use genetic or temperature dependent systems and even both simultaneously. The turtles are a particularly diverse group in the way they determine sex with multiple different genetic and temperature based systems having been described. We investigated the nature of the temperature based sex determination system in Trachemys scripta elegans to ascertain whether it behaved as a purely temperature based system or if some other global source of sex determining information might be apparent within thermal regions insufficient to fully induce male or female development. These experiments found that sex determination in this species is much more complex and early acting than previously thought and that each gonad within an individual has the same sexual fate established enough that it can persist even without further communication between. We established a best practice for the assembly and annotation of de novo whole transcriptomes from T. scripta RNA-seq and utilized the technique to quantify the gene regulatory events that occur across the thermal sensitive period.</p><p>Evidence is entirely lacking on the resolution of TSD when eggs are incubated at the pivitol temperature in which equal numbers or males and females are produces. We have produced a timecourse data set that allowed for the elucidation of the gene expression events that occur at both the MPT and FPT over the course of the thermal sensitive period. Our data suggests that early establishment of a male or female fate is possible when temperature is sufficiently strong enough as at MPT and FPT. We see a strong pattern of mutually antagonistic gene expression patterns emerging early and expanding over time through the end of the period of gonad plasticity. In addition, we have identified a strong pattern of differential expression in the early embryo at stages prior to the formation of the gonad. Even without the known systemic signaling attributed to sex hormones emanating from the gonad, the early embryo has a clear male and female gene expression pattern. We discuss how this early potential masculinization or feminization of the embryo may indicate that the influence of temperature may extend beyond the determination of gonadal sex or even metabolic adjustments and how this challenges the well-defined paradigm in which gonadal sex determines peripheral sexual characteristics.</p> / Dissertation

Seven families of XX and YY homozygous Oreochromis niloticus were produced by mitotic gynogenesis from XY neofemales and their genetic status was verified by multilocus DNA fingerprinting and progeny testing. Two of these gynogenetic families and their corresponding diploid controls were used with 64 AFLP primer combinations in different levels of screening (XX/YY grand pool; XX/YY family pool; XX/YY gynogenetics and XX/XY control individuals) to search for sex-linked or sex-specific markers. Grand pool screening did not reveal any sex-linked markers. Subsequent family pool and individual level screening identified four sex-linked AFLP markers from two primer combinations, three Y-linked (OniY425, OniY382, OniY227) and one X-linked (OniX420). Two of these (OniX420, OniY425) were shown to be allelic. Single locus PCR markers were developed for all of those markers. Linkage analysis of these markers and the sex locus within the source families revealed tight linkage, with estimated map distances of 13cM, 17cM and 20cM for OniY382, OniY227 and OniX420/OniY425 respectively. However, these sex-linked AFLP markers failed to consistently identify sex in unrelated individuals. To develop an effective system for parentage analysis in normal and gynogenetic progeny, AFLPs and multiplexed polymorphic microsatellite loci were investigated. Both were found to be effective, but microsatellites were more appropriate since they are codominant and some loci showed high gene-centromere recombination rates, suitable for discriminating meiotic from mitotic gynogenetics, while AFLPs are dominant markers. Spontaneous diploidization of the maternal chromosome set (SDM) was observed in gynogenetic progeny of one XY neofemale. Maternal inheritance and ploidy status were verified by multilocus DNA fingerprinting and chromosome karyotyping. Close genetic linkage between the red gene and an autosomal sex-reversal gene(s) in gynogenetic progeny and influences of autosomal sex-reversal gene(s) producing males in a fully inbred XX clonal line were previously reported in O. niloticus. To test if the same autosomal sex-reversal locus was responsible in both cases, a series of test crosses was carried out involving XX clonal neomale(s) and homozygous red females. The results indicated the involvement of more than one autosomal sex-reversal locus, one of which is linked to red body colour.

As moscas-das-frutas são consideras pragas da fruticultura mundial, sendo que a espécie de maior importância econômica na região Neotropical é Anastrepha fraterculus. Sabe-se que na verdade A. fraterculus corresponde a um complexo de espécies crípticas; vários estudos têm se focado na caracterização destas espécies e no desenvolvimento de estratégias de controle populacional. Trabalhos anteriores mostraram que o silenciamento de genes envolvidos na determinação sexual de Anastrepha, como o transformer-2, constitui uma potencial ferramenta que pode ser usada em programas com este propósito, por gerar uma maior proporção de machos, uma vez que a proteína Transformer-2 funcional é necessária para o desenvolvimento do fenótipo feminino. Na ausência desta proteína um embrião XX se desenvolve com o fenótipo masculino (pseudomacho). Esse tipo de abordagem pode ser utilizado para otimizar a produção de moscas em biofábricas. Nesses estudos prévios o dsRNA foi injetado diretamente em ovos, o que constitui uma técnica de execução bastante complexa e que resulta em uma alta mortalidade durante o processo, inviabilizando sua utilização em um contexto aplicado. Aqui nós descrevemos os efeitos do silenciamento gênico de transformer-2 a partir da injeção parental de dsRNA em A. sp.1 affinis fraterculus, com o intuito de verificar se ocorre o silenciamento gênico na progênie. Para isso, fêmeas adultas foram injetadas com transformer-2 dsRNA em seu abdome e cruzadas com machos não tratados. Suas progênies foram coletadas ao longo de cinco semanas e analisadas quanto a (a) proporção sexual, (b) presença de pseudomachos (XX), (c) fertilidade dos machos e (d) morfologia sexual, além de análises de (e) emergência e de (f) assimetria de estruturas dos adultos, que são indicadores relevantes para a aplicação dessa técnica. A análise da proporção sexual mostrou um desvio significativo da proporção esperada de 1?:1? a favor dos machos, sendo o máximo desvio detectado entre as semanas 1 e 3 após a injeção. A análise dos cariótipos revelou a presença de pseudomachos (XX) na progênie. A maioria dos adultos apresentou morfologia das estruturas sexuais normal, exceto um pequeno número de indivíduos, que tinham gônadas assimétricas. Os testes de fertilidade revelaram que todos os machos apresentaram espermatozoides móveis em seus testículos, mas algumas fêmeas, referentes aos cruzamentos estéreis, não tinham espermatozoides em suas espermatecas. As análises de emergência dos adultos e assimetria de asas e mostraram que não há diferenças em relação ao controle, mas há aumento da assimetria de cerdas frontais da cabeça. Esses resultados indicam que alguns indivíduos da progênie de fêmeas injetadas com transformer-2 dsRNA foram afetados pelo silenciamento gênico de transformer-2. Além disso, as análises de morfologia e assimetria sugerem que esse tratamento causa perturbações no desenvolvimento desses organismos. Finalmente, os testes de fertilidade mostraram que alguns machos da progênie são funcionalmente estéreis, por serem incapazes de transferir espermatozoides para as fêmeas, possivelmente os pseudomachos (XX). A produção de uma progênie com maior número de machos a partir da interferência na expressão de um único gene pode ser bastante útil para melhorar o manejo da produção de machos em biofábricas / Fruit flies are worldwide fruticulture pest, and the economic important species in Neotropics is Anastrepha fraterculus. It is known that actually A. fraterculus corresponds to a cryptic species complex; many studies have focused in characterizing those species and development of population control strategies. Previous studies showed that silencing genes involved in sex determination of Anastrepha, such as transformer-2, are potential tools to be used in control programs that require male production in a large scale. The Transformer-2 protein is required to development of female phenotype. In the absence of this protein, an XX embryo develops in male phenotype (pseudomale). In previous studies, dsRNA was injected directly in embryos, which consists in a high complex executing technique with high rates of mortality during this process, difficulting your utility in an applied context. Herein we describe the effects of transformer-2 gene silencing by parental injection of dsRNA in A. sp.1 affinis fraterculus, to verify whether gene silencing occurs in progeny. For that, adult females were injected with transformer-2 dsRNA in its abdomen and crossed to non-treated males. Their progeny were recovered by five weeks and analysed by (a) sex ratio, (b) presence of pseudomales (XX), (c) fertility of males and (d) sexual morphology, besides analysis of (e) emergence and (f) asymmetry of wings and head setae, what consists in indicators of goodness of progeny. Sex ratio analysis showed a significant bias of expected 1:1, in favour of males, significant between weeks 1 and 3. Karyotype analysis revealed presence of pseudomales (XX) in progeny. The most of flies exhibited sexual structures morphology normal, except by a few individuals having asymmetric testes. Fertility tests showed that all males had motile spermatozoids in their testes, but some females, regarding to sterile crosses, had no spermatozoids in their spermathecae. Emergence and wing asymmetry analysis showed that there is no significant differences in relation to control, but there is a significant increase in asymmetry of head setae. The results indicate that the progeny from females treated with transformer-2 dsRNA were affected by transformer-2 gene silencing. Besides, the morphological analysis suggest that the treatment has a low impact in disturbing ontogeny of the progeny comparing to method of injection directly in eggs. Finally, fertility tests showed that some males of progeny are functionally sterile, unable to transfer spermatozoids to females, possibly the pseudomales ones (XX). The production of a male enriched progeny by interference in expression of a single gene could be very useful to improve the management of male production in biofabrics

Florida’s sea turtle populations are increasing due to conservation efforts; however, sea turtle species are vulnerable to climate change. Turtles exhibit temperaturedependent sex determination, in which nest environment influences sex. Environmental changes may produce altered sex ratios that limit reproduction potential; therefore hatchling sex ratios should be monitored. Hatchlings are not externally sexually dimorphic, making sex identification difficult. This study established baseline plasma hormone concentrations in hatchling and post-hatchling green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtles using High Performance Liquid Chromatography. Five hormones were assayed and were present in the majority of samples (testosterone: N.D.-10.12, progesterone: N.D.-0.43, estradiol: N.D.-4.78, estriol: N.D.-5.55 and estrone: N.D.-1.67 μg/mL). Plasma hormones did not distinguish hatchling sex because male and female ranges overlapped. Hormone concentrations varied with sex but also with incubation temperature, indicating that climate change could impact hatchling and posthatchling hormone profiles and thus could impact future fitness. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

The identification of victims involved in mass fatality incidents, as well as the identification of unknown individuals in criminal cases has become an increasingly important issue nowadays. Sex assessment represents a key point in forensic evaluations due to its significance in providing biological identity. Even though the availability of documented skeletal remains to forensic practitioners is a common practice in many countries, in Turkey, contemporary documented skeletal remains are not available for this purpose. For this reason, studies have been focused on living populations. Previous research has shown that modern technologies such as CT scanning present very promising potential in establishing new standards for contemporary populations. Therefore, the main aim of this project was to examine the application of the measurements taken from 3D CT images of the femur in order to assess sex, and to contribute to the establishment of discriminant function equations for the Turkish population for forensic applications. The accuracy and reproducibility of imaging methods in the assessment of the measurements taken from femora are essential when estimating sex. This research also concentrated on determining the accuracy and repeatability of CT measurements, using the femur. Prior to primary data collection, a preliminary study was performed in an effort to test the reliability of the femur measurements. The results of reliability analysis indicated no significant difference between the three observations of each measurement. Thus, the methodology employed in the current study appears reliable and reproducible. In addition, a validation study was conducted to determine the linear measurement accuracy of the 3D volume rendering models derived from a medical CT scanner and the influence of different reconstruction parameters. The differences between measurements obtained from dry bones and their 3D volume rendered models were also evaluated. The results from this study indicated that there were no statistically significant differences between measurements taken from different reconstruction parameters and measurements obtained from CT images and drybones. Using the CT data, volume-rendering function (VR), 3D Curved Multiplanar reconstruction (MPR), and Scout View on OsiriX were employed in order to compare the accuracy and reliability of each rendering method and to determine which technique is optimal for linear measurements. Overall, the measurements taken from the 3D Volume Rendering images had the highest intra-observer reliability when compared to the other two rendering methods. This research study produced data and interpretations that will inform on and improve population specific standards of sex assessment from three-dimensional postcranial osteometric landmarks. Additionally, this research is believed to provide value for a developing discipline of forensic anthropology, and integrate within the existing systems of criminal investigation and disaster victim identification practices in Turkey. A Turkish sample population, consisting of 300 adult hospital patients was examined via the interpretation of CT reconstructed images using the OsiriX software. The 3D reconstructions were then created using the volume-rendering function in OsiriX (v.5.6.). Following the 3D reconstruction, an image of each femur was segmented from the surrounding bones to ensure the correct usage of landmarks as accurately as possible. Thirteen measurements were acquired using a 3D viewer after being located and marked on each CT reconstructed femora. These thirteen anthropometric parameters were measured and analysed by basic descriptive statistics and discriminant analysis methods using the SPSS 21.0 software package. The intra-observer variation was assessed by obtaining the intraclass correlation coefficient in order to evaluate the accuracy of the linear measurements taken. Asymmetry was also tested. The results indicated that an accuracy of 92.3% was acquired from a combination of six of the measurements, and the Femur Vertical Diameter of Neck (FVDN) measurement was found to be the most dimorphic with 88.0% accuracy.

The present research examined whether the innominate acted as contextual information which resulted in a skewed scoring of skull morphological sex traits and pairs overall (both innominate and skull). Survey participants first assessed the sex of an innominate, then assessed the sex of a skull, using methods standard in the field. The sex of ten skulls and associated innominates, which served as contextual elements to introduce bias, were assessed in two surveys by 22 participants total. Male and female innominates and skulls were mixed and matched to test bias, though it was implied to participants that the paired elements were from the same individual. No significant bias was seen on the level of the skull. However, significant shifts in scores were seen on the level of the overall pair in three out of the five studied, indicating that the sex of the innominate was privileged over that of the skull. This is considered standard procedure in the field as the innominate is more reliable for sex estimation; thus, the findings of the present study are inconsistent with cognitive bias. However, the present study raises questions about the utility of the skull in the estimation of sex in human skeletal remains. While the innominate may be more reliable in sex estimation when both elements are present, this may lead to inattention to valuable information presented by the skull in particular contexts such as commingled burials.

Submitted by William Justo Figueiro (williamjf) on 2015-07-20T23:20:31Z No. of bitstreams: 1 58c.pdf: 769092 bytes, checksum: d9e0dc98714105aed66b07b2534daa0b (MD5) / Made available in DSpace on 2015-07-20T23:20:31Z (GMT). No. of bitstreams: 1 58c.pdf: 769092 bytes, checksum: d9e0dc98714105aed66b07b2534daa0b (MD5) Previous issue date: 2012 / Nenhuma / Daption capense apresenta distribuição circumpolar, sendo bastante abundante no Hemisfério Sul. Reproduz principalmente em ilhas subantárticas, na Península e no Continente Antártico. Na Ilha Elefante, Arquipélago das Shetlands do Sul, também se apresenta como reprodutivo, porém poucos dados sobre a sua biologia são encontrados para esta localidade. É considerado um pequeno Procellariiforme, e dentro deste grupo, como a espécie que menos apresenta dimorfismo entre os indivíduos, porém podem apresentar certa diferença quanto às medidas morfométricas. Medidas morfométricas têm sido consideradas importantes ferramentas na suposição de determinado sexo. Considerando-se a importância das variáveis morfométricas na compreensão dos aspectos biológicos e em especial para a determinação sexual, objetivouse com este estudo (i) determinar o sexo dos indivíduos de D. capense através de técnicas moleculares e (ii) verificar quais variáveis morfométricas distinguem melhor machos de fêmeas. O estudo foi realizado na Ilha Elefante, nos períodos correspondentes ao verão austral, entre os anos de 2010/11 e 2011/12. Foram capturados 52 indivíduos adultos de D. capense, sendo 32 no verão austral de 2010/11 e 20 entre os anos de 2011/12, os quais foram anilhados e realizadas as medidas morfométricas. Foram coletadas ainda amostras de sangue para a realização de sexagem, dos quais 33 foram determinados como machos e 19 como fêmeas. As variáveis mais significativas para a determinação sexual, segundo a Análise Discriminante Stepwise foram dedo médio (F=12,222; gl 1,50; p=0,001) e a altura do bico (F=18,931; gl 2,49; / Daption capense presents circumpolar distribution, being very abundant in the Southern Hemisphere Plays mainly in subantarctic islands, the Antarctic Peninsula and on the Continent. In Elephant Island, South Shetland Archipelago is also presented as breeding, but few data on its biology are found for this location. It is considered a small Procellariiform, and within this group, as the species shows that less dimorphism among individuals, but may have some difference as to morphometric measurements. Given the importance of morphometric variables in the understanding of biological aspects, particularly for determining sexes, the objective of this study is to (i) determine the sex of individuals of D. capense through molecular techniques and (ii) verify which morphological variables better distinguish males from females. The study was conducted on Elephant Island, between October and March, which corresponds with austral summer in the Southern Hemisphere, in 2010/2011 and 2011/12. Fifty-two adult D. capense individuals were captured: 32 in the austral summer of 2010/11 and 20 in the years 2011/12. Each bird was banded and measured. Blood samples were collected to be used in determining the sex of the individuals, which showed that 33 were males and 19 were females. The average and standard error were calculated for the morphometric measurements of the individuals before and after sex determination. Sexual dimorphism between males and females of D. capense was confirmed, and the significant morphometric variables for sexual determination were middle toe length (F=12.222; gl 1, 50; p=0.001) and beak height (F=18.931; gl 2, 49; p

When unidentified skeletal remains are found, researchers utilize a number of methods to apportion details for a biological profile. While these practices are used and professed through generations of students, they also require a reevaluation of the methods. This project estimates the ancestry and sex of nine unknown skeletal individuals through two different mechanisms. Modified biological profiles were completed through two different methodologies: anthroscopic traits (Buikstra and Ubelaker 1994; White et al. 2012) and geometric morphometrics using 3D-ID (Slice and Ross 2009). The results serve two purposes: (1) to provide ancestry and sex (2) to compare two methodologies through outcomes and repeatability of results. Intra-observer error testing was conducted on both methods. All outputs resulted in low intra-rater reliability, highlighting the repeatability error in one observer’s collection methods. These results conclude and encourage the reevaluation and standardization of the procedures and comparison groups used to assess ancestry and sex. / Includes bibliography. / Thesis (M.A.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

Reptiles exhibit marked diversity in sex-determining mechanisms. Many species exhibit genotypic sex determination (GSD) with male heterogamety (XX females/XY males), others have GSD with female heterogamety (ZW females/ZZ males), and still others exhibit temperature-dependent sex determination (TSD). The distribution of these mechanisms throughout the reptile phylogeny implies evolutionary lability in sex determination, and in some lineages there has been a number of transitions between GSD and TSD. Despite this diversity, GSD and TSD have traditionally been viewed as mutually-exclusive mechanisms of sex determination in reptiles, since there is little evidence for their co-occurrence. Considerable empirical and theoretical effort has been directed towards understanding the adaptive significance of TSD in reptiles. In comparison, there has been little focus on understanding how evolutionary transitions between GSD and TSD occur at a genetic and mechanistic level. I addressed this question by applying both empirical and theoretical approaches to investigate interaction of genotypic and temperature influences in the sex determination of two endemic species of Australian lizards. The three-lined skink, Bassiana duperreyi, has XX/XY chromosomal sex determination, yet a previous investigation reported a significant male bias in the sex ratio of eggs incubated at low temperatures. To enable an explicit test for temperature induced sex reversal in this species, a 185 bp Y chromosome marker was isolated by Amplified Fragment Length Polymorphism (AFLP) analysis. The marker was subsequently converted into a duplex PCR assay that co-amplified a 185 bp (or 92 bp) Y chromosome fragment and a 356 bp fragment of the single-copy nuclear gene C-mos (from both sexes) as a positive control. The accuracy of the PCR sex assay was tested on 78 individuals for which sex reversal was not expected. PCR genotype and sex phenotype were concordant for 96% of the animals. This is one of the very few sex tests developed for a reptile, and the first report of Y chromosome sequence from a reptile. The PCR assay was subsequently applied to genotype hatchlings from both cool (16-7.5C) and warm (22-7.5C) cyclical incubation temperature treatments, and identified sex reversal in 15% of genotypically female (XX) embryos (n=26) from the cool treatment, but no sex reversal in eggs from the warmer treatment (n=35). Thus, low incubation temperatures can over-ride genotypic sex determination in B. duperreyi, indicating that GSD and TSD co-occur in this species. The Central bearded dragon, Pogona vitticeps (Agamidae), has ZZ/ZW chromosomal sex determination, and is a member of a lizard family in which GSD and TSD are both widespread, indicating evolutionary lability in sex determination. AFLP analysis was applied to isolate homologous Z and W chromosome-linked markers (71 bp and 72 bp, respectively) from this species. The AFLP sequences were subsequently extended into larger genomic fragments by a reiterated genome walking procedure, producing three non-overlapping contigs of 1.7 kb, 2.2 kb and 4.5 kb. The latter two fragments were verified as distinct, homologous Z/W chromosome fragments by PCR analyses. An amplified 3 kb fragment of the 4.5 kb contig was physically mapped to metaphase spreads, identifying the W microchromosome, and for the first time in this species, the Z microchromosome. PCR analyses indicated the presence of homologous sequences in other Australian agamid species, including both GSD and TSD species. The isolated sequences should therefore prove useful as a comparative genomic tool for investigating the genomic changes that have occurred in evolutionary transitions between sexdetermining mechanisms in agamids, by enabling the identification of chromosomes in TSD species that are homologous to the sex chromosomes of P. vitticeps. The isolated sequences were further converted into a duplex DNA sex assay that co-amplified a 224 bp W chromosome fragment and a 963 bp positive control fragment in both sexes. This PCR assay diagnosed chromosomal sex in three Pogona species, but was not effective outside the genus. Incubation treatment of P. vitticeps eggs revealed a strong and increasing female bias at high constant temperatures (34-36C), but an unbiased sex ratio between 22-32C. Hatchlings from three clutches split between 28C and 34 or 36C incubation treatments were genotyped with the W chromosome AFLP marker. At 28C, the sex ratio was 1:1 but the high temperature treatments produced 2 males and 33 females. All but one of the 30 lizards (97%) incubated at 28C had concordant sex phenotype and genotype, but only 18 of 35 animals (51%) from the high temperature treatment were concordant. All discordant animals were genotypic males (ZZ) that developed as females. Thus, temperature and genotypic influences can interact to determine sex in P. vitticeps. These empirical findings for B. duperreyi and P. vitticeps were extended into a novel theory for the evolution of sex-determining mechanisms in reptiles, working within the framework that species with temperature-induced reversal of chromosomal sex determination are a window to transitional stages of evolution between GSD and TSD. A model was derived from the observation that in both lizards, an extreme of incubation temperature causes sex reversal of the homogametic genotype. In this model, the strength of a genetic regulatory signal for sex determination must exceed a threshold for development of the homogametic sex to occur (male in Pogona, female in Bassiana). The strength of this signal is also temperature-sensitive, so diminishes at extremes of temperature. Simulation modelling demonstrated that increasing the relative magnitude of the threshold for sexual development can cause evolutionary transitions between GSD and TSD. Even more remarkably, decreasing the relative magnitude of the threshold value causes an evolutionary transition between female and male heterogametic GSD. Quantitative adjustment of a single model parameter (the threshold value) thus charts a continuous evolutionary pathway between the three principal mechanisms of sex determination in reptiles (XX/XY-ZZ/ZW-TSD), which were previously considered to be qualitatively distinct mechanisms. The experimental demonstration of temperature-induced reversal of chromosomal sex determination in both B. duperreyi and P. vitticeps presents a challenge to the traditional view that reptilian sex determination is strictly dichotomous (GSD or TSD), and suggests instead that sex determination in reptiles consists of a continuum of systems of interaction between genotypic and temperature influences. Simulation modelling provided solid theoretical support for this proposition, demonstrating that transitions along this continuum are effected simply through shifts in the mean population value for the sex-determining threshold, without requiring substantial genotypic innovation. An important implication of this theory is that transitions between XX/XY and ZZ/ZW modes of GSD may retain the same sex chromosome pair, and the same primary sexdetermining gene, in contrast to previous models for heterogametic transitions. A more immediate implication of these findings is that many reptile species believed to have strict TSD (in particular, lizards and crocodilians), may in fact have a sex-determining system of GSD-TSD interaction, where there is an equilibrium between GSD and TSD individuals within the population.