Sally R. Isberg

Three crocodilian genomes reveal ancestral patterns of evolution among archosaurs

INTRODUCTION Crocodilians and birds are the two extant clades of archosaurs, a group that includes the extinct dinosaurs and pterosaurs. Fossils suggest that living crocodilians (alligators, crocodiles, and gharials) have a most recent common ancestor 80 to 100 million years ago. Extant crocodilians are notable for their distinct morphology, limited intraspecific variation, and slow karyotype evolution. Despite their unique biology and phylogenetic position, little is known about genome evolution within crocodilians. Evolutionary rates of tetrapods inferred from DNA sequences anchored by ultraconserved elements. Evolutionary rates among reptiles vary, with especially low rates among extant crocodilians but high rates among squamates. We have reconstructed the genomes of the common ancestor of birds and of all archosaurs (shown in gray silhouette, although the morphology of these species is uncertain). RATIONALE Genome sequences for the American alligator, saltwater crocodile, and Indian gharial—representatives of all three extant crocodilian families—were obtained to facilitate better understanding of the unique biology of this group and provide a context for studying avian genome evolution. Sequence data from these three crocodilians and birds also allow reconstruction of the ancestral archosaurian genome. RESULTS We sequenced shotgun genomic libraries from each species and used a variety of assembly strategies to obtain draft genomes for these three crocodilians. The assembled scaffold N50 was highest for the alligator (508 kilobases). Using a panel of reptile genome sequences, we generated phylogenies that confirm the sister relationship between crocodiles and gharials, the relationship with birds as members of extant Archosauria, and the outgroup status of turtles relative to birds and crocodilians. We also estimated evolutionary rates along branches of the tetrapod phylogeny using two approaches: ultraconserved element–anchored sequences and fourfold degenerate sites within stringently filtered orthologous gene alignments. Both analyses indicate that the rates of base substitution along the crocodilian and turtle lineages are extremely low. Supporting observations were made for transposable element content and for gene family evolution. Analysis of whole-genome alignments across a panel of reptiles and mammals showed that the rate of accumulation of micro-insertions and microdeletions is proportionally lower in crocodilians, consistent with a single underlying cause of a reduced rate of evolutionary change rather than intrinsic differences in base repair machinery. We hypothesize that this single cause may be a consistently longer generation time over the evolutionary history of Crocodylia. Low heterozygosity was observed in each genome, consistent with previous analyses, including the Chinese alligator. Pairwise sequential Markov chain analysis of regional heterozygosity indicates that during glacial cycles of the Pleistocene, each species suffered reductions in effective population size. The reduction was especially strong for the American alligator, whose current range extends farthest into regions of temperate climates. CONCLUSION We used crocodilian, avian, and outgroup genomes to reconstruct 584 megabases of the archosaurian common ancestor genome and the genomes of key ancestral nodes. The estimated accuracy of the archosaurian genome reconstruction is 91% and is higher for conserved regions such as genes. The reconstructed genome can be improved by adding more crocodilian and avian genome assemblies and may provide a unique window to the genomes of extinct organisms such as dinosaurs and pterosaurs. 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.

Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes

The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described.

Comparative Genome Analyses Reveal Distinct Structure in the Saltwater Crocodile MHC

The major histocompatibility complex (MHC) is a dynamic genome region with an essential role in the adaptive immunity of vertebrates, especially antigen presentation. The MHC is generally divided into subregions (classes I, II and III) containing genes of similar function across species, but with different gene number and organisation. Crocodylia (crocodilians) are widely distributed and represent an evolutionary distinct group among higher vertebrates, but the genomic organisation of MHC within this lineage has been largely unexplored. Here, we studied the MHC region of the saltwater crocodile (Crocodylus porosus) and compared it with that of other taxa. We characterised genomic clusters encompassing MHC class I and class II genes in the saltwater crocodile based on sequencing of bacterial artificial chromosomes. Six gene clusters spanning ∼452 kb were identified to contain nine MHC class I genes, six MHC class II genes, three TAP genes, and a TRIM gene. These MHC class I and class II genes were in separate scaffold regions and were greater in length (2–6 times longer) than their counterparts in well-studied fowl B loci, suggesting that the compaction of avian MHC occurred after the crocodilian-avian split. Comparative analyses between the saltwater crocodile MHC and that from the alligator and gharial showed large syntenic areas (>80% identity) with similar gene order. Comparisons with other vertebrates showed that the saltwater crocodile had MHC class I genes located along with TAP, consistent with birds studied. Linkage between MHC class I and TRIM39 observed in the saltwater crocodile resembled MHC in eutherians compared, but absent in avian MHC, suggesting that the saltwater crocodile MHC appears to have gene organisation intermediate between these two lineages. These observations suggest that the structure of the saltwater crocodile MHC, and other crocodilians, can help determine the MHC that was present in the ancestors of archosaurs.

A genetic linkage map for the saltwater crocodile (Crocodylus porosus)

BackgroundGenome 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.ResultsA 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).ConclusionWe 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.

253 Novel polymorphic microsatellites for the saltwater crocodile (Crocodylus porosus)

Genomic elucidation and mapping of novel organisms requires the generation of large genetic resources. In this study, 253 novel and polymorphic microsatellite loci were isolated and characterized for the saltwater crocodile (Crocodylus porosus) by constructing libraries enriched for microsatellite DNA. All markers were evaluated on animals obtained from Darwin Crocodile Farm in the Northern Territory, Australia, and are intended for future use in the construction of a genetic-linkage map for the saltwater crocodile. The 253 loci yielded an average of 4.12 alleles per locus, and those selected for mapping had an average polymorphic information content (PIC) of 0.425.

Cross-species amplification of microsatellites in crocodilians: assessment and applications for the future

Microsatellite DNA loci have emerged as the dominant genetic tool for addressing questions associated with genetic diversity in many wildlife species, including crocodilians. Despite their usefulness, their isolation and development can be costly, as well as labour intensive, limiting their wider use in many crocodilian species. In this study, we investigate the cross-species amplification success of 82 existing microsatellites previously isolated for the saltwater crocodile (Crocodylus porosus) in 18 non-target crocodilian species; Alligator sinensis, Caiman crocodylus, Caiman latirostris, Caiman yacare, Melanosuchus niger, Paleosuchus palpebrosus, Crocodylus acutus, Mecistops cataphractus, Crocodylus intermedius, Crocodylus johnstoni, Crocodylus mindorensis, Crocodylus moreletii, Crocodylus niloticus, Crocodylus novaeguineae, Crocodylus palustis, Crocodylus rhombifer, Crocodylus siamensis, and Osteolaemus tetraspis. Our results show a high level of microsatellites cross-amplification making available polymorphic markers for a range of crocodilian species previously lacking informative genetic markers.

Reference levels for corticosterone and immune function in farmed saltwater crocodiles (Crocodylus porosus) hatchlings using current Code of Practice guidelines.

To determine reference levels for on-farm stressors on immune responsiveness and growth rate, 253 hatchling crocodiles from 11 known breeding pairs were repeatedly measured and blood sampled during their first year. Plasma corticosterone (CORT) was used to quantify baseline stress levels in captive animals and were found to be lower (mean 1.83±SE 0.16 ng/mL) than previously reported in saltwater crocodile hatchlings. Two tests of immune function were also conducted. Innate constitutive immunity was assessed using bacterial killing assays (BKA) against two bacterial species: Escherichia coli and Providencia rettgeri, whereby the latter causes considerable economic loss to industry from septicaemic mortalities. Although the bactericidal capabilities were different at approximately 4 months old (32±3% for E. coli and 16±4% for P. rettgeri), the differences had disappeared by approximately 9 months old (58±2% and 68±6%, respectively). To assess immune responsiveness to a novel antigen, the inflammatory swelling response caused by phytohaemagglutinin (PHA) injection was assessed but was only significantly different between Samplings 1 and 3 (5% LSD). There were no significant clutch effects for CORT or PHA but there were for both BKA traits. CORT was not significantly associated with growth (head length) or the immune parameters except for P. rettgeri BKA where higher CORT levels were associated with better bactericidal capability. As such, these results suggest that the crocodiles in this study are not stressed, therefore endorsing the management strategies adopted within the Australian industry Code of Practice.

Evolution and gene capture in ancient endogenous retroviruses - insights from the crocodilian genomes.

BackgroundCrocodilians 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.ResultsERVs 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.ConclusionsThis 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.

Using phytohaemagglutinin to determine immune responsiveness in saltwater crocodiles (Crocodylus porosus)

Abstract. Immune responsiveness, the ability of an organism to effectively respond immunologically following antigenic exposure, is an essential component of life history, as organisms require effective immune functionality in order to grow, survive and reproduce. However, immune status is also associated with concomitant trade-offs in these physiological functions. Herein we demonstrate the validation of phytohaemagglutinin (PHA) injection in saltwater crocodiles, Crocodylus porosus, to assess cellular immune responsiveness. Following injection of 2 mg mL–1 PHA into the hind toe webbing, we observed a peak swelling response 12 h after injection, with PHA inducing increased thickness compared with webs injected with phosphate-buffered saline (PBS) (F 5,518 = 145.13, P < 0.001). Subsequent injections increased responsiveness relative to the primary injection response (F 5,290 = 2.92, P = 0.029), suggesting that PHA exposure induced immunological memory, a tenet of acquired immunity. Histological examination revealed that PHA-injected toe webs displayed increased numbers of leukocytes (granulocytes, macrophages, and lymphocytes) relative to PBS-injected webs, with peak leukocytic infiltrate observed 12 h after injection. We suggest the use of PHA injection in crocodilians as a measure of cellular immune responsiveness in agricultural (production and animal welfare), ecological, and toxicological contexts.

Distribution of Endogenous Retroviruses in Crocodilians

ABSTRACT Knowledge of endogenous retroviruses (ERVs) in crocodilians (Crocodylia) is limited, and their distribution among extant species is unclear. Here we analyzed the phylogenetic relationships of these retroelements in 20 species of crocodilians by studying the pro-pol gene. The results showed that crocodilian ERVs (CERVs) cluster into two major clades (CERV 1 and CERV 2). CERV 1 clustered as a sister group of the genus Gammaretrovirus, while CERV 2 clustered distantly with respect to all known ERVs. Interestingly, CERV 1 was found only in crocodiles (Crocodylidae). The data generated here could assist future studies aimed at identifying orthologous and paralogous ERVs among crocodilians.