Daniel E. Janes

The genome of the green anole lizard and a comparative analysis with birds and mammals

The evolution of the amniotic egg was one of the great evolutionary innovations in the history of life, freeing vertebrates from an obligatory connection to water and thus permitting the conquest of terrestrial environments. Among amniotes, genome sequences are available for mammals and birds, but not for non-avian reptiles. Here we report the genome sequence of the North American green anole lizard, Anolis carolinensis. We find that A. carolinensis microchromosomes are highly syntenic with chicken microchromosomes, yet do not exhibit the high GC and low repeat content that are characteristic of avian microchromosomes. Also, A. carolinensis mobile elements are very young and diverse—more so than in any other sequenced amniote genome. The GC content of this lizard genome is also unusual in its homogeneity, unlike the regionally variable GC content found in mammals and birds. We describe and assign sequence to the previously unknown A. carolinensis X chromosome. Comparative gene analysis shows that amniote egg proteins have evolved significantly more rapidly than other proteins. An anole phylogeny resolves basal branches to illuminate the history of their repeated adaptive radiations.

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 genomics based on massive parallel transcriptome sequencing reveals patterns of substitution and selection across 10 bird species

Next‐generation sequencing technology provides an attractive means to obtain large‐scale sequence data necessary for comparative genomic analysis. To analyse the patterns of mutation rate variation and selection intensity across the avian genome, we performed brain transcriptome sequencing using Roche 454 technology of 10 different non‐model avian species. Contigs from de novo assemblies were aligned to the two available avian reference genomes, chicken and zebra finch. In total, we identified 6499 different genes across all 10 species, with ∼1000 genes found in each full run per species. We found evidence for a higher mutation rate of the Z chromosome than of autosomes (male‐biased mutation) and a negative correlation between the neutral substitution rate (dS) and chromosome size. Analyses of the mean dN/dS ratio (ω) of genes across chromosomes supported the Hill–Robertson effect (the effect of selection at linked loci) and point at stochastic problems with ω as an independent measure of selection. Overall, this study demonstrates the usefulness of next‐generation sequencing for obtaining genomic resources for comparative genomic analysis of non‐model organisms.

Sex Chromosome Evolution in Amniotes: Applications for Bacterial Artificial Chromosome Libraries

Variability among sex chromosome pairs in amniotes denotes a dynamic history. Since amniotes diverged from a common ancestor, their sex chromosome pairs and, more broadly, sex-determining mechanisms have changed reversibly and frequently. These changes have been studied and characterized through the use of many tools and experimental approaches but perhaps most effectively through applications for bacterial artificial chromosome (BAC) libraries. Individual BAC clones carry 100–200 kb of sequence from one individual of a target species that can be isolated by screening, mapped onto karyotypes, and sequenced. With these techniques, researchers have identified differences and similarities in sex chromosome content and organization across amniotes and have addressed hypotheses regarding the frequency and direction of past changes. Here, we review studies of sex chromosome evolution in amniotes and the ways in which the field of research has been affected by the advent of BAC libraries.

Evolution of sex chromosomes in Sauropsida

Reptiles (sauropsids) represent the sister group to mammals, and the basal members of Reptilia may provide a good model for the condition of the common ancestor of both groups. Sex-determining mechanisms (SDM) and organizations of sex chromosomes among genotypically sex-determining (GSD) species vary widely across reptiles. Birds and snakes, for example, are entirely GSD whereas other reptiles, like all crocodilians, exhibit temperature-dependent sex determination (TSD). Here we explore the evolution of sex chromosomes and SDM within reptiles, using family-level analyses of character evolution and applying parsimony, likelihood, Bayesian, and stochastic methods. We find support for the common ancestor of amphisbaenians and whiptail lizards (Laterata) possessing the XY (male heterogametic) GSD mechanism, while the ancestors of Testudines and Crocodylia, as well as the larger group Archosauromorpha (here containing turtles) are inferred to have exhibited TSD. We also find evidence consistent with the hypothesis that the XY system is more labile and evolves faster than does the ZW (female heterogametic) system. Phylogenetic-based speciation tests do not support an association between GSD and speciation, and reject the hypothesis that the presence of the XY system is associated with speciation in reptiles.

Genome Evolution in Reptilia, the Sister Group of Mammals

The genomes of birds and nonavian reptiles (Reptilia) are critical for understanding genome evolution in mammals and amniotes generally. Despite decades of study at the chromosomal and single-gene levels, and the evidence for great diversity in genome size, karyotype, and sex chromosome diversity, reptile genomes are virtually unknown in the comparative genomics era. The recent sequencing of the chicken and zebra finch genomes, in conjunction with genome scans and the online publication of the Anolis lizard genome, has begun to clarify the events leading from an ancestral amniote genome--predicted to be large and to possess a diverse repeat landscape on par with mammals and a birdlike sex chromosome system--to the small and highly streamlined genomes of birds. Reptilia exhibit a wide range of evolutionary rates of different subgenomes and, from isochores to mitochondrial DNA, provide a critical contrast to the genomic paradigms established in mammals.

Genotypic sex determination enabled adaptive radiations of extinct marine reptiles

Adaptive radiations often follow the evolution of key traits, such as the origin of the amniotic egg and the subsequent radiation of terrestrial vertebrates. The mechanism by which a species determines the sex of its offspring has been linked to critical ecological and life-history traits but not to major adaptive radiations, in part because sex-determining mechanisms do not fossilize. Here we establish a previously unknown coevolutionary relationship in 94 amniote species between sex-determining mechanism and whether a species bears live young or lays eggs. We use that relationship to predict the sex-determining mechanism in three independent lineages of extinct Mesozoic marine reptiles (mosasaurs, sauropterygians and ichthyosaurs), each of which is known from fossils to have evolved live birth. Our results indicate that each lineage evolved genotypic sex determination before acquiring live birth. This enabled their pelagic radiations, where the relatively stable temperatures of the open ocean constrain temperature-dependent sex determination in amniote species. Freed from the need to move and nest on land, extreme physical adaptations to a pelagic lifestyle evolved in each group, such as the fluked tails, dorsal fins and wing-shaped limbs of ichthyosaurs. With the inclusion of ichthyosaurs, mosasaurs and sauropterygians, genotypic sex determination is present in all known fully pelagic amniote groups (sea snakes, sirenians and cetaceans), suggesting that this mode of sex determination and the subsequent evolution of live birth are key traits required for marine adaptive radiations in amniote lineages.

Reptiles and mammals have differentially retained long conserved noncoding sequences from the amniote ancestor

Many noncoding regions of genomes appear to be essential to genome function. Conservation of large numbers of noncoding sequences has been reported repeatedly among mammals but not thus far among birds and reptiles. By searching genomes of chicken (Gallus gallus), zebra finch (Taeniopygia guttata), and green anole (Anolis carolinensis), we quantified the conservation among birds and reptiles and across amniotes of long, conserved noncoding sequences (LCNS), which we define as sequences ≥500 bp in length and exhibiting ≥95% similarity between species. We found 4,294 LCNS shared between chicken and zebra finch and 574 LCNS shared by the two birds and Anolis. The percent of genomes comprised by LCNS in the two birds (0.0024%) is notably higher than the percent in mammals (<0.0003% to <0.001%), differences that we show may be explained in part by differences in genome-wide substitution rates. We reconstruct a large number of LCNS for the amniote ancestor (ca. 8,630) and hypothesize differential loss and substantial turnover of these sites in descendent lineages. By contrast, we estimated a small role for recruitment of LCNS via acquisition of novel functions over time. Across amniotes, LCNS are significantly enriched with transcription factor binding sites for many developmental genes, and 2.9% of LCNS shared between the two birds show evidence of expression in brain expressed sequence tag databases. These results show that the rate of retention of LCNS from the amniote ancestor differs between mammals and Reptilia (including birds) and that this may reflect differing roles and constraints in gene regulation.

Parallel Evolution of Tetrodotoxin Resistance in Three Voltage-Gated Sodium Channel Genes in the Garter Snake Thamnophis sirtalis

Members of a gene family expressed in a single species often experience common selection pressures. Consequently, the molecular basis of complex adaptations may be expected to involve parallel evolutionary changes in multiple paralogs. Here, we use bacterial artificial chromosome library scans to investigate the evolution of the voltage-gated sodium channel (Nav) family in the garter snake Thamnophis sirtalis, a predator of highly toxic Taricha newts. Newts possess tetrodotoxin (TTX), which blocks Nav’s, arresting action potentials in nerves and muscle. Some Thamnophis populations have evolved resistance to extremely high levels of TTX. Previous work has identified amino acid sites in the skeletal muscle sodium channel Nav1.4 that confer resistance to TTX and vary across populations. We identify parallel evolution of TTX resistance in two additional Nav paralogs, Nav1.6 and 1.7, which are known to be expressed in the peripheral nervous system and should thus be exposed to ingested TTX. Each paralog contains at least one TTX-resistant substitution identical to a substitution previously identified in Nav1.4. These sites are fixed across populations, suggesting that the resistant peripheral nerves antedate resistant muscle. In contrast, three sodium channels expressed solely in the central nervous system (Nav1.1–1.3) showed no evidence of TTX resistance, consistent with protection from toxins by the blood–brain barrier. We also report the exon–intron structure of six Nav paralogs, the first such analysis for snake genes. Our results demonstrate that the molecular basis of adaptation may be both repeatable across members of a gene family and predictable based on functional considerations.

EVIDENCE FOR A GENOTYPE × ENVIRONMENT INTERACTION IN SEX-DETERMINING RESPONSE TO INCUBATION TEMPERATURE IN THE LEOPARD GECKO, EUBLEPHARIS MACULARIUS

Among vertebrates, sex is determined by environmental or genotypic sex determination, such that sex is determined by incubation environment or by the genetic contribution of parents, respectively. The selective advantage of one mechanism over the other is unclear and our understanding of the evolution of environmental sex determination (ESD) is obscured by the lack of description of the mechanisms genetic architecture. In this study, a reptile with environmental sex determination, the leopard gecko, Eublepharis macularius, was tested for a genotype × environment interaction (G×E) with respect to sex-determining response to incubation temperature. Five sires were each mated to five unique dams. The eggs from each sire/dam combination were randomly assigned to one of three incubation temperatures and resultant hatchling sex ratios were measured. Temperature and temperature × dam (sire) interaction significantly affected treatment group sex. We report a statistically significant effect of genotype by environment interaction (G×E) on the offspring sex in E. macularius. G×E for ESD may provide an opportunity for evolution for different threshold conditions for sex determination in this ESD species.