BackgroundWe describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing.ResultsOur phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented.ConclusionsOur comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.

Since its initial discovery in a Drosophila mutagenesis screen, the Hedgehog pathway has been revealed to be instrumental in the proper development of the vertebrate face. Vertebrates possess three hedgehog paralogs: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Of the three, Shh has the broadest range of functions both in the face and elsewhere in the embryo, while Ihh and Dhh play more limited roles. The Hedgehog pathway is instrumental from the period of prechordal plate formation early in the embryo, until the fusion of the lip and secondary palate, which complete the major patterning events of the face. Disruption of Hedgehog signaling results in an array of developmental disorders in the face, ranging from minor alterations in the distance between the eyes to more serious conditions such as severe clefting of the lip and palate. Despite its critical role, Hedgehog signaling seems to be disrupted through a number of mechanisms that may either be direct, as in mutation of a downstream target of the Hedgehog ligand, or indirect, such as mutation in a ciliary protein that is otherwise seemingly unrelated to the Hedgehog pathway. A number of teratogens such as alcohol, statins and steroidal alkaloids also disrupt key aspects of Hedgehog signal transduction, leading to developmental defects that are similar, if not identical, to those of Hedgehog pathway mutations. The aim of this review is to highlight the variety of roles that Hedgehog signaling plays in developmental disorders of the vertebrate face.

The amniote primary palate encompasses the upper lip and the nasal cavities. During embryonic development, the primary palate forms from the fusion of the maxillary, medial nasal and lateral nasal prominences. In mammals, as the primary palate fuses, the nasal and oral cavities become completely separated. Subsequently, the tissue demarcating the future internal nares (choanae) thins and becomes the bucconasal membrane, which eventually ruptures and allows for the essential connection of the oral and nasal cavities to form. In reptiles (including birds), the other major amniote group, primary palate ontogeny is poorly studied with respect to prominence fusion, especially the formation of a bucconasal membrane. Using 3D optical projection tomography, we found that the prominences that initiate primary palate formation are similar between mammals and crocodilians but distinct from turtles and lizards, which are in turn similar to each other. Chickens are distinct from all non-avian lineages and instead resemble human embryos in this aspect. The majority of reptiles maintain a communication between the oral and nasal cavities via the choanae during primary palate formation. However, crocodiles appear to have a transient separation between the oral and nasal cavities. Furthermore, the three lizard species examined here, exhibit temporary closure of their external nares via fusion of the lateral nasal prominences with the frontonasal mass, subsequently reopening them just before hatching. The mechanism of the persistent choanal opening was examined in chicken embryos. The mesenchyme posterior/dorsal to the choana had a significant decline in proliferation index, whereas the mesenchyme of the facial processes remained high. This differential proliferation allows the choana to form a channel between the oral and nasal cavities as the facial prominences grow and fuse around it. Our data show that primary palate ontogeny has been modified extensively to support the array of morphological diversity that has evolved among amniotes.

The assembly of the upper jaw is a pivotal moment in the embryonic development of amniotes. The upper jaw forms from the fusion of the maxillary, medial nasal, and lateral nasal prominences, resulting in an intact upper lip/beak and nasal cavities; together called the primary palate. This process of fusion requires a balance of proper facial prominence shape and positioning to avoid craniofacial clefting, whilst still accommodating the vast phenotypic diversity of adult amniotes. As such, variation in craniofacial ontogeny is not tolerated beyond certain bounds. For clarity, we discuss primary palatogenesis of amniotes into in two categories, according to whether the nasal and oral cavities remain connected throughout ontogeny or not. The transient separation of these cavities occurs in mammals and crocodilians, while remaining connected in birds, turtles and squamates. In the latter group, the craniofacial prominences fuse around a persistent choanal groove that connects the nasal and oral cavities. Subsequently, all lineages except for turtles, develop a secondary palate that ultimately completely or partially separates oral and nasal cavities. Here, we review the shared, early developmental events and highlight the points at which development diverges in both primary and secondary palate formation.

Batrachochytrium dendrobatidis is the causative agent of chytridiomycosis, which is considered one of the driving forces behind the worldwide decline in populations of amphibians. As a member of the phylum Chytridiomycota, B. dendrobatidis has diverged significantly to emerge as the only pathogen of adult vertebrates. Such shifts in lifestyle are generally accompanied by various degrees of genomic modifications, yet neither its mode of pathogenicity nor any factors associated with it have ever been identified. Presented here is the identification and characterization of a unique expansion of the carbohydrate-binding module family 18 (CBM18), specific to B. dendrobatidis. CBM (chitin-binding module) expansions have been likened to the evolution of pathogenicity in a variety of fungus species, making this expanded group a prime candidate for the identification of potential pathogenicity factors. Furthermore, the CBM18 expansions are confined to three categories of genes, each having been previously implicated in host-pathogen interactions. These correlations highlight this specific domain expansion as a potential key player in the mode of pathogenicity in this unique fungus. The expansion of CBM18 in B. dendrobatidis is exceptional in its size and diversity compared to other pathogenic species of fungi, making this genomic feature unique in an evolutionary context as well as in pathogenicity.

The development of the reproductive system in bufonids (true toads) is unique in several respects: sexual differentiation occurs later than in other anurans, and toads develop a Bidder's organ, a rudimentary ovary that can be manipulated in males to produce mature oocytes. To illuminate the genesis of this unusual reproductive system, we isolated from the cane toad (Bufo marinus) the orthologues of several known vertebrate sex-determining genes, determined their primary structure, and studied their expression by reverse transcriptase-polymerase chain reaction and in situ hybridization of tissue sections. We report here that cane toad Sox9, Dmrt1, and p450aromatase (Cyp19a1) are highly homologous to their counterparts in other vertebrates. They show profiles of expression that generally follow patterns observed in other taxa, but with some novel features. Our data suggest that these genes likely play key roles in sex determination and early gonad development in bufonids.

During embryonic development, amniotes typically form outgrowths from the medial sides of the maxillary prominences called palatal shelves or palatine processes. In mammals the shelves fuse in the midline and form a bony hard palate that completely separates the nasal and oral cavities. In birds and lizards, palatine processes develop but remain unfused, leaving a natural cleft. Adult turtles do not possess palatine processes and unlike other amniotes, the internal nares open into the oral cavity. Here we investigate craniofacial ontogeny in the turtle, Emydura subglobosa to determine whether vestigial palatine processes develop and subsequently regress, or whether development fails entirely. We found that the primary palate in turtles develops similarly to other amniotes, but secondary palate ontogeny diverges. Using histology, cellular dynamics and in situ hybridization we found no evidence of palatine process development at any time during ontogeny of the face in the turtle. Furthermore, detailed comparisons with chicken embryos (the model organism most closely related to turtles from a molecular phylogeny perspective), we identified differences in proliferation and gene expression patterns that correlate with the differences in palate morphology. We propose that, in turtles, palatine process outgrowth is never initiated due to a lack of mesenchymal bone morphogenetic protein 2 (BMP2) expression in the maxillary mesenchyme, which in turn fails to induce the relatively higher cellular proliferation required for medial tissue outgrowth. It is likely that these differences between turtles and birds arose after the divergence of the lineage leading to modern turtles.

The cane toad (Bufo marinus) is one of the most notorious animal pests encountered in Australia. Members of the genus Bufo historically have been regarded as having genotypic sex determination with male homogamety/female heterogamety. Nevertheless, as with many toads, karyotypic analyses of the cane toad have so far failed to identify heteromorphics sex chromosomes. In this study, we used comparative genomic hybridization, reverse fluorescence staining, C-banding, and morphometric analyses of chromosomes to characterize sex chromosome dimorphism in B. marinus. We found that females consistently had a length dimorphism associated with a nucleolus organizer region (NOR) on one of the chromosome 7 pair. A strong signal over the longer NOR in females, and the absence of a signal in males indicated sex-specific DNA sequences. All females were heterozygous and all males homozygous, indicating a ZZ/ZW sex chromosomal system. Our study confirms the existence of sex chromosomes in this species. The ability to reliably identify genotypic sex of cane toads will be of value in monitoring and control efforts in Australia and abroad.