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The epidermis of amniotes forms a protective barrier against the environment and the differentiation program of keratinocytes, the main cell type in the epidermis, has undergone specific alterations in the course of adaptation of amniotes to a broad variety of environments and lifestyles. The epidermal differentiation complex (EDC) is a cluster of genes expressed at late stages of keratinocyte differentiation in both sauropsids and mammals. In the present study, we identified and analyzed the crocodilian equivalent of the EDC. The gene complement of the EDC of both the American alligator and the saltwater crocodile were determined by comparative genomics, de novo gene prediction and identification of EDC transcripts in published transcriptome data. We found that crocodilians have an organization of the EDC similar to that of their closest living relatives, the birds, with which they form the clade Archosauria. Notable differences include the specific expansion of a subfamily of EDC genes in crocodilians and the loss of distinct ancestral EDC genes in birds. Identification and comparative analysis of crocodilian orthologs of avian feather proteins suggest that the latter evolved by cooption and sequence modification of ancestral EDC genes, and that the amplification of an internal highly cysteine-enriched amino acid sequence motif gave rise to the feather component epidermal differentiation cysteine-rich protein in the avian lineage. Thus, sequence diversification of EDC genes contributed to the evolutionary divergence of the crocodilian and avian integuments. [ABSTRACT FROM AUTHOR]

We deduced the amino acid (aa) sequence of the nuclear factor kappa B (NFκB) protein from genomic data for the American alligator ( Alligator mississippiensis ), the estuarine crocodile ( Crocodylus porosus ), and the Indian gharial ( Gavialis gangeticus ). A 105 kDa protein, NFκB1 exhibits complex post-translational processing, multiple mechanisms of activation, and acts as precursor for a p50, a Rel homology transcription factor which influences the expression of key genes for developmental processes, apoptosis, and immune function. The aa sequences of the crocodylian proteins share very high identity with each other (97.2 ± 0.7%), birds (81.0 ± 1.1%, n = 6), mammals (75.3 ± 1.6%, n = 4), reptiles (80.3 ± 5.1%, n = 2), and less identity with fish (55.5 ± 5.5%, n = 4) and one amphibian (66.1 ± 0.8%). The crocodylian protein has a well-conserved Rel homology domain, a nuclear localization signal, and a glycine-rich region which facilitates proteasome-mediated generation of p50. The Rel homology domain contains sequences responsible for dimerization, DNA-binding, and nuclear translocation. In addition, seven ankyrin repeats were located, which putatively allow for inhibition of transcriptional regulation by mediating interaction with Inhibitor kappa B. Other features include a death domain, and conserved serine residues, near the C-terminal end, which act as potential phosphorylation sites for activation of the proteolytic generation of p50. Western blot analysis showed both the 105 kDa precursor and the 50 kDa mature NFκB were expressed in the alligator liver. Nuclear factor κB exhibited diffuse cytoplasmic distribution in alligator hepatocytes, and almost no cytoplasmic localization in infected animals. In addition, nuclear NFκB exhibited specific binding to the consensus NFκB promoter element. [ABSTRACT FROM AUTHOR]

Maintaining genetic diversity is crucial for the survival and management of threatened and endangered species. In this study, we analyzed genetic diversity and population genetic structure at neutral loci in American crocodiles, Crocodylus acutus, from several areas (Parque Nacional Marino Las Baulas, Parque Nacional Santa Rosa, Parque Nacional Palo Verde, Rio Tarcoles and Osa Conservation Area) in Pacific Costa Rica. We genotyped 184 individuals at nine microsatellite loci to describe the genetic diversity and conservation genetics between and among populations. No population was at Hardy-Weinberg Equilibrium (HWE) over all loci tested and a small to moderate amount of inbreeding was present. Populations along the Pacific coast had an average heterozygosity of 0.572 across all loci. All populations were significantly differentiated from each other with both FST and RST measures of population differentiation with a greater degree of molecular variance (81%) found within populations. Our results suggest C. acutus populations in Pacific Costa Rica were not panmictic with moderate levels of genetic diversity. An effective management plan that maintains the connectivity between clusters is critical to the success of C. acutus in Pacific Costa Rica. [ABSTRACT FROM AUTHOR]

Recent genetic and morphological evidence indicates that the African dwarf crocodile ( O. tetraspis) is comprised of three highly divergent lineages: O. sp. nov., O. tetraspis and O. osborni. Their putative distributional limits correspond with the Cameroon volcanic line (CVL) and the Congo River basin. In this study, we expanded on previous phylogeographic work by conducting detailed sampling of this crocodile in Cameroon and around the CVL. We tested whether O. tetraspis is the only lineage occurring in Cameroon and whether the CVL represents the distributional limit between O. sp. nov. and O. tetraspis. We collected 65 tissue samples from individuals located throughout Cameroon, and the eastern and western sides of the CVL. We sequenced fragments of two mitochondrial genes (CO1 and 12S rDNA) and one nuclear gene (LDH-A). We found that O. tetraspis extends west beyond the CVL and, thus, this mountain chain does not represent the distributional limit of this lineage. We also found O. osborni in Cameroon. Our findings have important implications for the conservation and management of O. tetraspis lineages. [ABSTRACT FROM AUTHOR]

Background Crocodilians are thought to be hosts to a diverse and divergent complement of endogenous retroviruses (ERVs) but a comprehensive investigation is yet to be performed. The recent sequencing of three crocodilian genomes provides an opportunity for a more detailed and accurate representation of the ERV diversity that is present in these species. Here we investigate the diversity, distribution and evolution of ERVs from the genomes of three key crocodilian species, and outline the key processes driving crocodilian ERV proliferation and evolution. Results ERVs and ERV related sequences make up less than 2% of crocodilian genomes. We recovered and described 45 ERV groups within the three crocodilian genomes, many of which are species specific. We have also revealed a new class of ERV, ERV4, which appears to be common to crocodilians and turtles, and currently has no characterised exogenous counterpart. For the first time, we formally describe the characteristics of this ERV class and its classification relative to other recognised ERV and retroviral classes. This class shares some sequence similarity and sequence characteristics with ERV3, although it is phylogenetically distinct from the other ERV classes. We have also identified two instances of gene capture by crocodilian ERVs, one of which, the capture of a host KIT-ligand mRNA has occurred without the loss of an ERV domain. Conclusions This study indicates that crocodilian ERVs comprise a wide variety of lineages, many of which appear to reflect ancient infections. In particular, ERV4 appears to have a limited host range, with current data suggesting that it is confined to crocodilians and some lineages of turtles. Also of interest are two ERV groups that demonstrate evidence of host gene capture. This study provides a framework to facilitate further studies into non-mammalian vertebrates and highlights the need for further studies into such species. [ABSTRACT FROM AUTHOR]

To provide context for the diversification of archosaurs--the group that includes crocodilians, dinosaurs, and birds--we generated draft genomes of three crocodilians: Alligator mississippiensis (the American alligator), Crocodylus porosus (the saltwater crocodile), and Gavialis gangeticus (the Indian gharial). We observed an exceptionally slow rate of genome evolution within crocodilians at all levels, including nucleotide substitutions, indels, transposable element content and movement, gene family evolution, and chromosomal synteny. When placed within the context of related taxa including birds and turtles, this suggests that the common ancestor of all of these taxa also exhibited slow genome evolution and that the comparatively rapid evolution is derived in birds. The data also provided the opportunity to analyze heterozygosity in crocodilians, which indicates a likely reduction in population size for all three taxa through the Pleistocene. Finally, these data combined with newly published bird genomes allowed us to reconstruct the partial genome of the common ancestor of archosaurs, thereby providing a tool to investigate the genetic starting material of crocodilians, birds, and dinosaurs. [ABSTRACT FROM AUTHOR]

The W chromosome of ratite birds shows minimal morphological differentiation and retains homology of genetic linkage and gene order with a substantial stretch of the Z chromosome; however, the molecular structure in the differentiated region is still not well known. The kW1 sequence was isolated from the kiwi as a W-specific DNA marker for PCR-based molecular sexing of ratite birds. In ratite W chromosomes, this sequence commonly contains a ∼200-bp deletion. To characterize the very early event of avian sex chromosome differentiation, we performed molecular cytogenetic analyses of kW1 and its flanking sequences in paleognathous and neognathous birds and reptiles. Female-specific repeats were found in the kW1-flanking sequence of the cassowary (Casuarius casuarius), and the repeats have been amplified in the pericentromeric region of the W chromosomes of ratites, which may have resulted from the cessation of meiotic recombination between the Z and W chromosomes at an early stage of sex chromosome differentiation. The presence of the kW1 sequence in neognathous birds and a crocodilian species suggests that the kW1 sequence was present in the ancestral genome of Archosauria; however, it disappeared in other reptilian taxa and several lineages of neognathous birds. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Major Histocompatibility Complex (MHC) class II genes encode for molecules that aid in the presentation of antigens to helper T cells. MHC characterisation within and between major vertebrate taxa has shed light on the evolutionary mechanisms shaping the diversity within this genomic region, though little characterisation has been performed within the Order Crocodylia. Here we investigate the extent and effect of selective pressures and trans-species polymorphism on MHC class II α and β evolution among 20 extant species of Crocodylia. Selection detection analyses showed that diversifying selection influenced MHC class II β diversity, whilst diversity within MHC class II α is the result of strong purifying selection. Comparison of translated sequences between species revealed the presence of twelve trans-species polymorphisms, some of which appear to be specific to the genera Crocodylus and Caiman. Phylogenetic reconstruction clustered MHC class II α sequences into two major clades representing the families Crocodilidae and Alligatoridae. However, no further subdivision within these clades was evident and, based on the observation that most MHC class II α sequences shared the same trans-species polymorphisms, it is possible that they correspond to the same gene lineage across species. In contrast, phylogenetic analyses of MHC class II β sequences showed a mixture of subclades containing sequences from Crocodilidae and/or Alligatoridae, illustrating orthologous relationships among those genes. Interestingly, two of the subclades containing sequences from both Crocodilidae and Alligatoridae shared specific trans-species polymorphisms, suggesting that they may belong to ancient lineages pre-dating the divergence of these two families from the common ancestor 85–90 million years ago. The results presented herein provide an immunogenetic resource that may be used to further assess MHC diversity and functionality in Crocodylia. [ABSTRACT FROM AUTHOR]

The archosauria consist of two living groups, crocodilians, and birds. Here we compare the structure, expression, and phylogeny of the beta (ß)-keratins in two crocodilian genomes and two avian genomes to gain a better understanding of the evolutionary origin of the feather ß-keratins. Unlike squamates such as the green anole with 40 ß-keratins in its genome, the chicken and zebra finch genomes have over 100 ß-keratin genes in their genomes, while the American alligator has 20 ß-keratin genes, and the saltwater crocodile has 21 ß-keratin genes. The crocodilian ß-keratins are similar to those of birds and these structural proteins have a central filament domain and N- and C-termini, which contribute to the matrix material between the twisted ß-sheets, which form the 2-3 nm filament. Overall the expression of alligator ß-keratin genes in the integument increases during development. Phylogenetic analysis demonstrates that a crocodilian ß-keratin clade forms a monophyletic group with the avian scale and feather ß-keratins, suggesting that avian scale and feather ß-keratins along with a subset of crocodilian ß-keratins evolved from a common ancestral gene/s. Overall, our analyses support the view that the epidermal appendages of basal archosaurs used a diverse array of ß-keratins, which evolved into crocodilian and avian specific clades. In birds, the scale and feather subfamilies appear to have evolved independently in the avian lineage from a subset of archosaurian claw ß-keratins. The expansion of the avian specific feather ß-keratin genes accompanied the diversification of birds and the evolution of feathers. [ABSTRACT FROM AUTHOR]

Background: Genome elucidation is now in high gear for many organisms, and whilst genetic maps have been developed for a broad array of species, surprisingly, no such maps exist for a crocodilian, or indeed any other non-avian member of the Class Reptilia. Genetic linkage maps are essential tools for the mapping and dissection of complex quantitative trait loci (QTL), and in order to permit systematic genome scans for the identification of genes affecting economically important traits in farmed crocodilians, a comprehensive genetic linage map will be necessary. Results: A first-generation genetic linkage map for the saltwater crocodile (Crocodylus porosus) was constructed using 203 microsatellite markers amplified across a two-generation pedigree comprising ten full-sib families from a commercial population at Darwin Crocodile Farm, Northern Territory, Australia. Linkage analyses identified fourteen linkage groups comprising a total of 180 loci, with 23 loci remaining unlinked. Markers were ordered within linkage groups employing a heuristic approach using CRIMAP v3.0 software. The estimated female and male recombination map lengths were 1824.1 and 319.0 centimorgans (cM) respectively, revealing an uncommonly large disparity in recombination map lengths between sexes (ratio of 5.7:1). Conclusion: We have generated the first genetic linkage map for a crocodilian, or indeed any other non-avian reptile. The uncommonly large disparity in recombination map lengths confirms previous preliminary evidence of major differences in sex-specific recombination rates in a species that exhibits temperature-dependent sex determination (TSD). However, at this point the reason for this disparity in saltwater crocodiles remains unclear. This map will be a valuable resource for crocodilian researchers, facilitating the systematic genome scans necessary for identifying genes affecting complex traits of economic importance in the crocodile industry. In addition, since many of the markers placed on this genetic map have been evaluated in up to 18 other extant species of crocodilian, this map will be of intrinsic value to comparative mapping efforts aimed at understanding genome content and organization among crocodilians, as well as the molecular evolution of reptilian and other amniote genomes. As researchers continue to work towards elucidation of the crocodilian genome, this first generation map lays the groundwork for more detailed mapping investigations, as well as providing a valuable scaffold for future genome sequence assembly. [ABSTRACT FROM AUTHOR]

Chicken repeat 1 (CR1) retroposons are the most abundant superfamily of transposable elements in the genomes of birds, crocodilians, and turtles. However, CR1 mobilization remains poorly understood. In this article, I document that the diverse CR1 lineages of land vertebrates share a highly conserved hairpin structure and an octamer microsatellite motif at their very 3′ ends. Together with the presence of these same motifs in the tails of CR1-mobilized short interspersed elements, this suggests that the minimum requirement for CR1 transcript recognition and retrotransposition is a complex >50-nt structure. Such a highly specific recognition sequence readily explains why CR1-dominated genomes generally contain very few retrogenes. Conversely, the mammalian richness in retrogenes results from CR1 extinction in their early evolution and subsequent establishment of L1 dominance. [ABSTRACT FROM AUTHOR]

Accurate species delimitation is a central assumption of biology that, in groups such as the Crocodylia, is often hindered by highly conserved morphology and frequent introgression. In Africa, crocodilian systematics has been hampered by complex regional biogeography and confounded taxonomic history. We used rigorous molecular and morphological species delimitation methods to test the hypothesis that the slender-snouted crocodile (Mecistops cataphractus) is composed of multiple species corresponding to the Congolian and Guinean biogeographic zones. Speciation probability was assessed by using 11 mitochondrial and nuclear genes, and cranial morphology for over 100 specimens, representing the full geographical extent of the species distribution. Molecular Bayesian and phylogenetic species delimitation showed unanimous support for two Mecistops species isolated to the Upper Guinean and Congo (including Lower Guinean) biomes that were supported by 13 cranial characters capable of unambiguously diagnosing each species. Fossil-calibrated phylogenetic reconstruction estimated that the species split ± 6.5-7.5 Ma, which is congruent with intraspecies divergence within the sympatric crocodile genus Osteolaemus and the formation of the Cameroon Volcanic Line. Our results underscore the necessity of comprehensive phylogeographic analyses within currently recognized taxa to detect cryptic species within the Crocodylia. We recommend that the community of crocodilian researchers reconsider the conceptualization of crocodilian species especially in the light of the conservation ramifications for this economically and ecologically important group. [ABSTRACT FROM AUTHOR]