Partial Sex Reversal Research Articles

The Chinese medaka (Oryzias sinensis) dmrt1 gene converts females to males in medaka (Oryzias latipes)

BackgroundChinese medaka (Oryzias sinensis) is widely distributed in freshwater rivers in China. Similar to the medaka (Oryzias latipes), Chinese medaka has the characteristics of small size, rapid reproductive cycle, and strong adaptability, which makes it suitable as a model organism for studies in basic biology and environmental toxicology. Chinese medaka exhibits distinct sexual dimorphism. However, due to the lack of complete genomic information, the regulation of sex determination and differentiation-related genes in Chinese medaka remains unclear. MethodsChinese medaka dmrt1 (Osdmrt1) was cloned by PCR, and transgenic individuals of medaka [Tg(CMV:Osdmrt1)] overexpressing Osdmrt1 were generated to investigate the role of Osdmrt1 in sex determination. Western blot was used to validate the integration of the Osdmrt1 into the medaka genome. Tissue sectioning and HE staining were used to identify Tg(CMV:Osdmrt1) physiological gender and phenotype. qRT-PCR was used to analyze the expression of gonad-specific genes. ResultsOsdmrt1 was cloned and identified, and it shared similar evolutionary relationships with medaka dmrt1. Tg(CMV:Osdmrt1) exhibited partial sex reversal from female to male in the F2 generation, with genetically female individuals developing testes and producing functional sperm. Additionally, the secondary sexual characteristics of the transgenic females also changed to males. ConclusionThe Chinese medaka dmrt1 gene could convert females to males in medaka.General significance: These results not only elucidate the function of Chinese medaka dmrt1, but also accumulate knowledge for studying the function of economically important fish genes in model fish by transgenic technology.

Knockdown of R-spondin1 leads to partial sex reversal in genetic female Chinese soft-shelled turtle Pelodiscus sinensis

Chinese soft-shelled turtle Pelodiscus sinensis is an important aquaculture species in China, the male individual being more valuable in aquaculture because of its larger body size, higher growth rate and less fat compared with females. Understanding the mechanism of ovarian differentiation and development is crucial for the production of mono-sex male offspring. However, little is known about the molecular mechanism underlying turtle ovarian differentiation. Here, we characterized the Rspo1 gene, an upstream regulator of vertebrate female sexual differentiation, in P. sinensis. The messenger RNA of Rspo1 was initially expressed at stage 14, preceding gonadal sex differentiation, and exhibited a sexually dimorphic expression pattern throughout the sex determination and gonadal differentiation periods. Rspo1 was rapidly downregulated during aromatase inhibitor-induced female-to-male sex reversal, which occurred prior to gonadal differentiation. Rspo1 loss of function by RNA interference led to partial female-to-male sex reversal, with masculinized changes in the phenotype of gonads, the distribution of germ cells and the expression of testicular regulators. Collectively, these findings suggest that Rspo1 is necessary for primary female sexual differentiation in P. sinensis. This study demonstrates for the first time the functional role of Rspo1 in reptilian sex determination, and is of fundamental significance for the production of fertile pseudo-female parents and mono-sex male offspring of P.sinensis.

Heterozygous deletion of Sox9 in mouse mimics the gonadal sex reversal phenotype associated with campomelic dysplasia in humans.

Heterozygous mutations in the human SOX9 gene cause the skeletal malformation syndrome campomelic dysplasia which in 75% of 46, XY individuals is associated with male-to-female sex reversal. Although studies in homozygous Sox9 knockout mouse models confirmed that SOX9 is critical for testis development, mice heterozygous for the Sox9-null allele were reported to develop normal testes. This led to the belief that the SOX9 dosage requirement for testis differentiation is different between humans, which often require both alleles, and mice, in which one allele is sufficient. However, in prior studies, gonadal phenotypes in heterozygous Sox9 XY mice were assessed only by either gross morphology, histological staining or analyzed on a mixed genetic background. In this study, we conditionally inactivated Sox9 in somatic cells of developing gonads using the Nr5a1-Cre mouse line on a pure C57BL/6 genetic background. Section and whole-mount immunofluorescence for testicular and ovarian markers showed that XY Sox9 heterozygous gonads developed as ovotestes. Quantitative droplet digital PCR confirmed a 50% reduction of Sox9 mRNA as well as partial sex reversal shown by an upregulation of ovarian genes. Our data show that haploinsufficiency of Sox9 can perturb testis development in mice, suggesting that mice may provide a more accurate model of human disorders/differences of sex development than previously thought.

Gtsf1 is essential for proper female sex determination and transposon silencing in the silkworm, Bombyx mori.

Sex determination pathways are astoundingly diverse in insects. For instance, the silk moth Bombyx mori uniquely use various components of the piRNA pathway to produce the Fem signal for specification of the female fate. In this study, we identified BmGTSF1 as a novel piRNA factor which participates in B. mori sex determination. We found that BmGtsf1 has a distinct expression pattern compared to Drosophila and mouse. CRISPR/Cas9 induced mutation in BmGtsf1 resulted in partial sex reversal in genotypically female animals by shifting expression of the downstream targets BmMasc and Bmdsx to the male pattern. As levels of Fem piRNAs were substantially reduced in female mutants, we concluded that BmGtsf1 plays a critical role in the biogenesis of the feminizing signal. We also demonstrated that BmGTSF1 physically interacted with BmSIWI, a protein previously reported to be involved in female sex determination, indicating BmGTSF1 function as the cofactor of BmSIWI. BmGtsf1 mutation resulted in piRNA pathway dysregulation, including piRNA biogenesis defects and transposon derepression, suggesting BmGtsf1 is also a piRNA factor in the silkworm. Furthermore, we found that BmGtsf1 mutation leads to gametogenesis defects in both male and female. Our data suggested that BmGtsf1 is a new component involved in the sex determination pathway in B. mori.

Molecular and genetic characterization of partial masculinization in embryonic ovaries grafted into male nude mice.

In most of mammalian embryos, gonadal sex differentiation occurs inside the maternal uterus before birth. In several fetal ovarian grafting experiments using male host mice, an experimental switch from the maternal intrauterine to male-host environment gradually induces partial masculinization of the grafted ovaries even under the wild-type genotype. However, either host-derived factors causing or molecular basis underlying this masculinization of the fetal ovaries are not clear. Here, we demonstrate that ectopic appearance of SOX9-positive Sertoli cell-like cells in grafted ovaries was mediated by the testosterone derived from the male host. Neither Sox8 nor Amh activity in the ovarian tissues is essential for such ectopic appearance of SOX9-positive cells. The transcriptome analyses of the grafted ovaries during this masculinization process showed early downregulation of pro-ovarian genes such as Irx3, Nr0b1/Dax1, Emx2, and Fez1/Lzts1 by days 7–10 post-transplantation, and subsequent upregulation of several pro-testis genes, such as Bhlhe40, Egr1/2, Nr4a2, and Zc3h12c by day 20, leading to a partial sex reversal with altered expression profiles in one-third of the total numbers of the sex-dimorphic pre-granulosa and Sertoli cell-specific genes at 12.5 dpc. Our data imply that the paternal testosterone exposure is partially responsible for the sex-reversal expression profiles of certain pro-ovarian and pro-testis genes in the fetal ovaries in a temporally dependent manner.

Multiplexed CRISPR/Cas9-mediated knockout of 19 Fanconi anemia pathway genes in zebrafish revealed their roles in growth, sexual development and fertility.

Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp-/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway.

Loss of p300 and CBP disrupts histone acetylation at the mouse Sry promoter and causes XY gonadal sex reversal.

CREB-binding protein (CBP, CREBBP, KAT3A) and its closely related paralogue p300 (EP300, KAT3B), together termed p300/CBP, are histone/lysine acetyl-transferases that control gene expression by modifying chromatin-associated proteins. Here, we report roles for both of these chromatin-modifying enzymes in mouse sex determination, the process by which the embryonic gonad develops into a testis or an ovary. By targeting gene ablation to embryonic gonadal somatic cells using an inducible Cre line, we show that gonads lacking either gene exhibit major abnormalities of XY gonad development at 14.5 dpc, including partial sex reversal. Embryos lacking three out of four functional copies of p300/Cbp exhibit complete XY gonadal sex reversal and have greatly reduced expression of the key testis-determining genes Sry and Sox9. An analysis of histone acetylation at the Sry promoter in mutant gonads at 11.5 dpc shows a reduction in levels of the positive histone mark H3K27Ac. Our data suggest a role for CBP/p300 in testis determination mediated by control of histone acetylation at the Sry locus and reveal a novel element in the epigenetic control of Sry and mammalian sex determination. They also suggest possible novel causes of human disorders of sex development (DSD).

Sequential, Divergent, and Cooperative Requirements of Foxl2a and Foxl2b in Ovary Development and Maintenance of Zebrafish.

Foxl2 is essential for mammalian ovary maintenance. Although sexually dimorphic expression of foxl2 was observed in many teleosts, its role and regulative mechanism in fish remained largely unclear. In this study, we first identified two transcript variants of foxl2a and its homologous gene foxl2b in zebrafish, and revealed their specific expression in follicular layer cells in a sequential and divergent fashion during ovary differentiation, maturation, and maintenance. Then, homozygous foxl2a mutants (foxl2a-/-) and foxl2b mutants (foxl2b-/-) were constructed and detailed comparisons, such as sex ratio, gonadal histological structure, transcriptome profiling, and dynamic expression of gonadal development-related genes, were carried out. Initial ovarian differentiation and oocyte development occur normally both in foxl2a-/- and foxl2b-/- mutants, but foxl2a and foxl2b disruptions result in premature ovarian failure and partial sex reversal, respectively, in adult females. In foxl2a-/- female mutants, sox9a-amh/cyp19a1a signaling was upregulated at 150 days postfertilization (dpf) and subsequently oocyte apoptosis was triggered after 180 dpf. In contrast, dmrt1 expression was greater at 105 dpf and increased several 100-fold in foxl2b-/- mutated ovaries at 270 dpf, along with other testis-related genes. Finally, homozygous foxl2a-/-/foxl2b-/- double mutants were constructed in which complete sex reversal occurs early and testis-differentiation genes robustly increase at 60 dpf. Given mutual compensation between foxl2a and foxl2b in foxl2b-/- and foxl2a-/- mutants, we proposed a model in which foxl2a and foxl2b cooperate to regulate zebrafish ovary development and maintenance, with foxl2b potentially having a dominant role in preventing the ovary from differentiating as testis, as compared to foxl2a.

Fungal Infection Induces Sex-Specific Transcriptional Changes and Alters Sexual Dimorphism in the Dioecious Plant Silene latifolia.

Sexual dimorphism, including differences in morphology, behavior and physiology between females and males, is widespread in animals and plants and is shaped by gene expression differences between the sexes. Such expression differences may also underlie sex-specific responses of hosts to pathogen infections, most notably when pathogens induce partial sex reversal in infected hosts. The genetic changes associated with sex-specific responses to pathogen infections on the one hand, and sexual dimorphism on the other hand, remain poorly understood. The dioecious White Campion (Silene latifolia) displays sexual dimorphism in floral traits and infection with the smut fungus Micobrotryum lychnidis-dioicae induces a partial sex reversal in females. We find strong sex-specific responses to pathogen infection and reduced sexual dimorphism in infected S. latifolia. This provides a direct link between pathogen-mediated changes in sex-biased gene expression and altered sexual dimorphism in the host. Expression changes following infection affected mainly genes with male-biased expression in healthy plants. In females, these genes were up-regulated, leading to a masculinization of the transcriptome. In contrast, infection in males was associated with down-regulation of these genes, leading to a demasculinization of the transcriptome. To a lesser extent, genes with female-biased expression in healthy plants were also affected in opposite directions in the two sexes. These genes were overall down-regulated in females and up-regulated in males, causing, respectively, a defeminization in infected females and a feminization of the transcriptome in infected males. Our results reveal strong sex-specific responses to pathogen infection in a dioecious plant and provide a link between pathogen-induced changes in sex-biased gene expression and sexual dimorphism.

Antagonistic Roles of Dmrt1 and Foxl2 in Sex Differentiation via Estrogen Production in Tilapia as Demonstrated by TALENs

Transcription activator-like effector nucleases (TALENs) are a powerful approach for targeted genome editing and have been proved to be effective in several organisms. In this study, we reported that TALENs can induce somatic mutations in Nile tilapia, an important species for worldwide aquaculture, with reliably high efficiency. Six pairs of TALENs were constructed to target genes related to sex differentiation, including dmrt1, foxl2, cyp19a1a, gsdf, igf3, and nrob1b, and all resulted in indel mutations with maximum efficiencies of up to 81% at the targeted loci. Effects of dmrt1 and foxl2 mutation on gonadal phenotype, sex differentiation, and related gene expression were analyzed by histology, immunohistochemistry, and real-time PCR. In Dmrt1-deficient testes, phenotypes of significant testicular regression, including deformed efferent ducts, degenerated spermatogonia or even a complete loss of germ cells, and proliferation of steroidogenic cells, were observed. In addition, disruption of Dmrt1 in XY fish resulted in increased foxl2 and cyp19a1a expression and serum estradiol-17β and 11-ketotestosterone levels. On the contrary, deficiency of Foxl2 in XX fish exhibited varying degrees of oocyte degeneration and significantly decreased aromatase gene expression and serum estradiol-17β levels. Some Foxl2-deficient fish even exhibited complete sex reversal with high expression of Dmrt1 and Cyp11b2. Furthermore, disruption of Cyp19a1a in XX fish led to partial sex reversal with Dmrt1 and Cyp11b2 expression. Taken together, our data demonstrated that TALENs are an effective tool for targeted gene editing in tilapia genome. Foxl2 and Dmrt1 play antagonistic roles in sex differentiation in Nile tilapia via regulating cyp19a1a expression and estrogen production.

Conserved action of β-catenin during female fate determination in the red-eared slider turtle.

In reptiles such as the red-eared slider turtle Trachemys scripta, development of an ovary from the bipotential gonad requires a coordinated expansion of the cortical domain and regression of the medulla. While estrogen, which is necessary and sufficient for ovarian development in non-mammalian vertebrates, is thought to feminize both compartments, it remains unclear whether there is a signaling relationship between the two domains that coordinates their fates. We show that aromatase, the estrogen-synthesizing enzyme, is localized to the medulla of the differentiating turtle ovary and that differentiation of the medulla precedes and is independent of cortical expansion. Coordinated feminization of the two domains may therefore rely on an estrogenic signal from the differentiating medulla. In eutherian mammals, where estrogen is dispensable for early ovary development, the canonical Wnt signaling pathway is critical for female fate determination. Whether this function is conserved among vertebrates and how it is potentially integrated with estrogen signaling are uncertain. Using a novel in vitro turtle gonad culture system, we demonstrate that ectopic activation of the canonical Wnt signaling pathway in presumptive male gonads induced a partial sex-reversal of the medulla, but inhibition of the pathway was not sufficient to sex-reverse differentiating ovaries. These patterns are similar to those previously observed in mice. Wnt signaling appears to function downstream of estrogen, as ectopic activation of the pathway rescued female development when estrogen synthesis was inhibited. Our findings therefore suggest that the ovary-promoting effects of the Wnt signaling pathway may be functionally conserved between mammals and reptiles.

Generation of an allele to inactivate Wnt4 gene function conditionally in the mouse

The Wnt family member Wnt4 is critical for the development of several organs, including the kidney, gonad, and adrenal, mammary, and pituitary glands. To study its potential postnatal functions, we generated a floxed Wnt4 allele. A single loxP site was targeted to the second intron, while a loxP-Neo-loxP cassette was placed 3' from Exon 5. The floxed Neo cassette was subsequently removed by crossing with MeuCre40 transgenic mice. The Wnt4 gene was specifically inactivated with CAGCre and another Wnt4 allele, Wnt4(EGFPCre), in which the Cre is driven by the endogenous Wnt4 promoter. Deletion of Wnt4 gene function with CAGCre impaired kidney development, as is the case with the conventional knockout. Similarly, the Wnt4(EGFPCre)-mediated inactivation of Wnt4 function considerably reduced the amount of Wnt4 transcripts, led to a severe defect in kidney development, and caused the female embryos to undergo partial sex reversal to males. All in all, the floxed Wnt4 allele serves as a useful tool for studying the roles of Wnt4 signaling during the life cycle.

The avian Z-linked gene DMRT1 is required for male sex determination in the chicken

Sex in birds is chromosomally based, as in mammals, but the sex chromosomes are different and the mechanism of avian sex determination has been a long-standing mystery. In the chicken and all other birds, the homogametic sex is male (ZZ) and the heterogametic sex is female (ZW). Two hypotheses have been proposed for the mechanism of avian sex determination. The W (female) chromosome may carry a dominant-acting ovary determinant. Alternatively, the dosage of a Z-linked gene may mediate sex determination, two doses being required for male development (ZZ). A strong candidate avian sex-determinant under the dosage hypothesis is the conserved Z-linked gene, DMRT1 (doublesex and mab-3-related transcription factor 1). Here we used RNA interference (RNAi) to knock down DMRT1 in early chicken embryos. Reduction of DMRT1 protein expression in ovo leads to feminization of the embryonic gonads in genetically male (ZZ) embryos. Affected males show partial sex reversal, characterized by feminization of the gonads. The feminized left gonad shows female-like histology, disorganized testis cords and a decline in the testicular marker, SOX9. The ovarian marker, aromatase, is ectopically activated. The feminized right gonad shows a more variable loss of DMRT1 and ectopic aromatase activation, suggesting differential sensitivity to DMRT1 between left and right gonads. Germ cells also show a female pattern of distribution in the feminized male gonads. These results indicate that DMRT1 is required for testis determination in the chicken. Our data support the Z dosage hypothesis for avian sex determination.

Effects of Embryonic Treatment with Fadrozole on Phenotype of Gonads, Syrinx, and Neural Song System in Zebra Finches

Previous studies have found that treatment of zebra finch embryos with an aromatase inhibitor on Day 5 or 8 of incubation caused partial sex reversal of gonadal phenotype in females. These females possessed both testicular and ovarian tissue, and the development of the neural circuit for song remained feminine. The present study attempted more complete gonadal reversal by treating zebra finch embryos earlier, on Day 3 of incubation, with Fadrozole (CGS 16949A), an aromatase inhibitor, or with saline. We examined the phenotype of the syrinx (androgen-dependent vocal organ), the gonads, and the telencephallic neural song system in 100-day-old birds. Treated females typically possessed a left ovotestis and a right testis, and significantly larger syringes than control females. The histology and steroid synthetic enzyme activity of the testicular tissue in treated females were quite masculine and similar to that of control males. At the time of sacrifice, the plasma concentrations of testosterone and estradiol for fadrozole-treated females did not differ from those of control females, but dihydrotestosterone was lower in treated females. Despite the large amount of functional testicular tissue and a masculine syrinx, the volumes and soma sizes of song system nuclei (HVC, RA) in treated females remained feminine. These results suggest that testicular secretions masculinize the syrinx, but are not sufficient to masculinize the song system in zebra finches.

An autosomal or X linked mutation results in true hermaphrodites and 46,XX males in the same family.

It is now well established that the differentiation of the primitive gonad into the testis during early human embryonic development depends on the presence of the SRY gene. However, the...

Sex determination in dioecious Silene latifolia. Effects of the Y chromosome and the parasitic smut fungus (Ustilago violacea) on gene expression during flower development.

We have embarked on a molecular cloning approach to the investigation of sex determination in Silene latifolia Poiret, a dioecious plant species with morphologically distinguishable sex chromosomes. One of our key objectives was to define a range of genes that are up-regulated in male plants in response to Y chromosome sex-determination genes. Here we present the characterization of eight male-specific cDNA sequences and classify these according to their expression dynamics to provide a range of molecular markers for dioecious male flower development. Genetically female S. latifolia plants undergo a partial sex reversal in response to infection by the parasitic smut fungus Ustilago violacea. This phenomenon has been exploited in these studies; male-specific cDNAs have been further categorized as inducible or noninducible in female plants by smut fungus infection. Analysis of the organ-specific expression of male-specific probes in male and female flowers has also identified a gene that is regulated in a sex-specific manner in nonreproductive floral tissues common to both male and female plants. This observation provides, to our knowledge, the first molecular marker for dominant effect of the Y chromosome in nonreproductive floral organs.

Evidence to exclude SOX9 as a candidate gene for XY sex reversal without skeletal malformation.

The skeletal malformation syndrome campomelic dysplasia (CMD1) is caused by mutations within the SOX9 gene or chromosomal rearrangement breakpoints outside SOX9. Approximately three quarters of cases of CMD1 in XY...

Spontaneous sex reversal inPleurodeles waltl(urodele amphibia): analysis of its inheritance

SummaryUp to now, no case of spontaneous sex reversal has been described in the urodele amphibianPleurodeles waltlreared at ambient temperature. However in offspring reared under laboratory conditions, males, females but also intersex individuals were obtained. The males, some females and the intersexes had the ZZ male genotype identified through a test performed with a sex-linked enzyme. The other females had the ZW female genotype. Using the animals of this particular strain, an offspring analysis was made on crosses between, respectively, ZZ males and ZW females, ZZ males and ZZ females, and ZZ intersexes and ZZ males or females. All these crosses gave ZZ males, ZZ females and ZZ intersexes. The spontaneous complete or partial sex reversal is inheritable, but the genetic mechanism of this inheritance has not yet been elucidated.

Assays of testis development in the mouse distinguish three classes of domesticus-type Y chromosome.

The Y chromosome of Mus musculus poschiavinus interacts with the autosomal recessive gene tda-1b of the C57BL/6J laboratory strain of the house mouse to cause complete or partial sex reversal. Ovaries or ovotestes develop in a substantial proportion of the XY fetuses. Several different Y-specific DNA probes distinguish two major types of Y chromosome in the house mouse and they are represented by M. m. domesticus and M. m. musculus. The poschiavinus Y chromosome appears identical to the domesticus Y. The developmental distribution of the gonad types was examined in the first backcross or N2 generation of fetuses in C57BL/6J with six different domesticus-type Y chromosomes and, as controls, three different musculus-type Y chromosomes. Gonadal hermaphrodites were found with three of the six domesticus-type Y chromosomes. Both overall frequency and phenotypic distribution of types of gonadal hermaphrodites identify three classes of domesticus-type Y chromosome by their differential interaction with the C57BL/6J genetic background.

Angiosperm phylogeny, floral morphology and pollination ecology

The different aspects of floral evolution—Angiosperm descent, floral morphology and pollination ecology—are discussed on the basis of the anthocorm theory of the angiospermous flower. Opposed ideas are critically compared and rejected mainly on account of several inconsistencies and flaws in old floral concepts. Floral evolution passed from a very early phase of dicliny, anemophily and aphananthy of the anthocorm to a phase of incipient entomophily soon associated with a partial sex reversal within the anthocorm. This second phase culminated in the monoclinous and primarily zoophilous flower types, whereas other floral types, more particularly the primarily anemophilous, diclinous flowers, are the direct descendants of the anthocorms of the initial phase. The second phase (to be differentiated in phase II and phase IIA) gave rise to at least two morphologically different flower types and two separate semophyletic lines of floral evolution (phase III and III A), in each of which the primary zoophily is almost always associated with the development of intrafloral semaphylls (petals) usually (at least in dicotyledonous groups) of androecial derivation. Early flower types were aphananthous and clearly anthocormoid (a condition still approximately represented by the so-called inflorescences of theSaururaceae) and assumed the morphology and other characteristics of a true flower by contractions, condensations, readjustments, oligomerisations, reductions and modifications (semaphylls!) leading to the great variety of adaptive floral patterns.