John W. Finger

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.

Adapterama I: Universal stubs and primers for thousands of dual-indexed Illumina libraries (iTru & iNext)

Next-generation DNA sequencing (NGS) offers many benefits, but major factors limiting NGS include reducing the time and costs associated with: 1) start-up (i.e., doing NGS for the first time), 2) buy-in (i.e., getting any data from a run), and 3) sample preparation. Although many researchers have focused on reducing sample preparation costs, few have addressed the first two problems. Here, we present iTru and iNext, dual-indexing systems for Illumina libraries that help address all three of these issues. By breaking the library construction process into re-usable, combinatorial components, we achieve low start-up, buy-in, and per-sample costs, while simultaneously increasing the number of samples that can be combined within a single run. We accomplish this by extending the Illumina TruSeq dual-indexing approach from 20 (8+12) indexed adapters that produce 96 (8x12) unique combinations to 579 (192+387) indexed primers that produce 74,304 (192x387) unique combinations. We synthesized 208 of these indexed primers for validation, and 206 of them passed our validation criteria (99% success). We also used the indexed primers to create hundreds of libraries in a variety of scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter which works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: 1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and 2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Although the methods we present are highly customizable, resulting libraries can be used with the standard Illumina sequencing primers and demultiplexed with the standard Illumina software packages, thereby minimizing instrument and software customization headaches. In subsequent Adapterama papers, we use these same iTru primers with different adapter stubs to construct double- to quadruple-indexed amplicon libraries and double-digest restriction-site associated DNA (RAD) libraries. For additional details and updates, please see http://baddna.org.Next-generation DNA sequencing (NGS) offers many benefits, but major factors limiting NGS include reducing costs of: 1) start-up (i.e., doing NGS for the first time); 2) buy-in (i.e., getting the smallest possible amount of data from a run); and 3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: 1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and 2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent Adapterama papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms.

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.

Endocrine-disrupting chemical exposure and the American alligator: a review of the potential role of environmental estrogens on the immune system of a top trophic carnivore.

Endocrine-disrupting chemicals (EDCs) alter cellular and organ system homeostasis by interfering with the body’s normal physiologic processes. Numerous studies have identified environmental estrogens as modulators of EDC-related processes in crocodilians, notably in sex determination. Other broader studies have shown that environmental estrogens dysregulate normal immune function in mammals, birds, turtles, lizards, fish, and invertebrates; however, the effects of such estrogenic exposures on alligator immune function have not been elucidated. Alligators occupy a top trophic status, which may give them untapped utility as indicators of environmental quality. Environmental estrogens are also prevalent in the waters they occupy. Understanding the effects of these EDCs on alligator immunity is critical for managing and assessing changes in their health and is thus the focus of this review.

Evaluating the effect of sample type on American alligator (Alligator mississippiensis) analyte values in a point-of-care blood analyser

Point-of-care blood analyzers provide researchers with the opportunity to collect blood biochemistry data for a variety of non-domesticed organisms while in the field. We investigated the use of an i-STAT analyzer and CG4+ cartridge to analyze whole blood, fresh plasma, and previously frozen plasma samples collected from American alligators.

IN OVO AND IN VITRO SUSCEPTIBILITY OF AMERICAN ALLIGATORS (ALLIGATOR MISSISSIPPIENSIS) TO AVIAN INFLUENZA VIRUS INFECTION

Abstract Avian influenza has emerged as one of the most ubiquitous viruses within our biosphere. Wild aquatic birds are believed to be the primary reservoir of all influenza viruses; however, the spillover of H5N1 highly pathogenic avian influenza (HPAI) and the recent swine-origin pandemic H1N1 viruses have sparked increased interest in identifying and understanding which and how many species can be infected. Moreover, novel influenza virus sequences were recently isolated from New World bats. Crocodilians have a slow rate of molecular evolution and are the sister group to birds; thus they are a logical reptilian group to explore susceptibility to influenza virus infection and they provide a link between birds and mammals. A primary American alligator (Alligator mississippiensis) cell line, and embryos, were infected with four, low pathogenic avian influenza (LPAI) strains to assess susceptibility to infection. Embryonated alligator eggs supported virus replication, as evidenced by the influenza virus M gene and infectious virus detected in allantoic fluid and by virus antigen staining in embryo tissues. Primary alligator cells were also inoculated with the LPAI viruses and showed susceptibility based upon antigen staining; however, the requirement for trypsin to support replication in cell culture limited replication. To assess influenza virus replication in culture, primary alligator cells were inoculated with H1N1 human influenza or H5N1 HPAI viruses that replicate independent of trypsin. Both viruses replicated efficiently in culture, even at the 30 C temperature preferred by the alligator cells. This research demonstrates the ability of wild-type influenza viruses to infect and replicate within two crocodilian substrates and suggests the need for further research to assess crocodilians as a species potentially susceptible to influenza virus infection.

Effects of selenium exposure on the hematology, innate immunity, and metabolic rate of yellow-bellied sliders ( Trachemys scripta scripta )

Selenium (Se) is a naturally occurring essential element that can be toxic to vertebrates at high concentrations. Despite studies that have documented that wild reptile species can accumulate copious amounts of Se, little is known regarding specific toxicologic effects of Se. In this study, 70 juvenile yellow-bellied sliders (Trachemys scripta scripta) were exposed to one of three seleno-l-methionine (SetMet) treatments (control, n = 24; 15 mg/kg, n = 23; and 30 mg/kg, n = 23) via weekly oral gavage for 5 weeks. At the conclusion of the experiment, kidney, liver, muscle, and blood samples were collected for quantitative Se analysis. Turtles in the SeMet treatment groups accumulated significantly higher amounts of Se in all tissue types relative to controls (all p < 0.001). Turtles in the 30 mg/kg SeMet group also accumulated significantly higher amounts of Se compared to the 15 mg/kg group (all p < 0.001). Although toxicity thresholds for reptiles have not been established, Se concentrations in liver tissue from both SeMet treatment groups exceeded reported avian toxicity thresholds for liver tissue. Neither oxygen consumption nor innate bactericidal capacity were impacted by SeMet exposure. However, turtles in the 30 mg/kg SeMet group exhibited anemia, which has been reported in other vertebrates exposed to Se. Furthermore, juvenile T. s. scripta in the 30 mg/kg SeMet group experienced 17% mortality compared to 0% in the 15 mg/kg treatment and control groups. To our knowledge, this study is the first to report dose-dependent Se-associated anemia and mortality in a chelonian species.

Early stress priming and the effects on fitness‐related traits following an adult stress exposure

Developmental stressors can have strong effects that persist well into adulthood, and are generally seen as detrimental. However, recent work suggests that a mild developmental stressor can have beneficial effects by preparing the organism to better withstand negative impacts when exposed to high levels of the stressor later in life, also known as a conditioning hormesis. Still, little is known about the influence of such hormetic effects on fitness-related measures. We hypothesized that exposure to a mild stressor during development will protect individuals later in life from the negative effects of a high heat stressor on immune function and reproduction. To test this hypothesis, we subjected zebra finches (Taeniopygia guttata) to a repeated mild heat stressor (38°C) as juveniles for 28 days. As adults, the birds were then exposed to a high heat stressor (42°C) for 3 consecutive days and we examined the effects on immune function via wound healing, and on female reproductive output. We found that females given the mild heat stressor as juveniles healed wounds marginally slower, but also had higher clutch viability than controls. For the adult treatment, we saw that high heat had a stimulatory effect on clutch viability as well. Our findings point toward the occurrence of trade-offs between immune function and reproduction due to a cost of hormetic priming when the adult environment does not match that of early life.

Adapterama III: Quadruple-indexed, triple-enzyme RADseq libraries for about

Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially due to the inability to highly-multiplex samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that relies on adapters and primers designed in Adapterama I. Major features of our method include: 1) minimizing the number of processing steps; 2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; 3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; 4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; 5) interchangeable adapter designs; 6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; 7) maintaining compatibility with universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, 8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.Molecular ecologists have used genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from individuals being studied (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently. Here, we present a low-cost and highly robust approach for the construction of dual-digest RADseq libraries. Major features of our method include: 1) minimizing the number of processing steps; 2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; 3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; 4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter thus increasing the efficiency of adapter ligation to sample DNA; 5) integrating adapter designs that can be used interchangeably; 6) incorporating variable-length internal tags within the adapters to increase the scope of sample tagging and facilitate pooling while also increasing sequence diversity; 7) maintaining compatibility with universal dual-indexed primers, which facilitate construction of combinatorial quadruple-indexed libraries that are compatible with standard Illumina sequencing reagents and libraries; and, 8) easy tuning for molecular tagging and PCR duplicate identification. We present eight adapter designs that work with 72 restriction enzyme combinations, and we demonstrate our approach by the use of one set of adapters and one set of restriction enzymes across a variety of non-model organisms to discover thousands of variable loci in each species.