Drew R. Schield

The Burmese python genome reveals the molecular basis for extreme adaptation in snakes

Significance The molecular basis of morphological and physiological adaptations in snakes is largely unknown. Here, we study these phenotypes using the genome of the Burmese python (Python molurus bivittatus), a model for extreme phenotypic plasticity and metabolic adaptation. We discovered massive rapid changes in gene expression that coordinate major changes in organ size and function after feeding. Many significantly responsive genes are associated with metabolism, development, and mammalian diseases. A striking number of genes experienced positive selection in ancestral snakes. Such genes were related to metabolism, development, lungs, eyes, heart, kidney, and skeletal structure—all highly modified features in snakes. Snake phenotypic novelty seems to be driven by the system-wide coordination of protein adaptation, gene expression, and changes in genome structure. Snakes possess many extreme morphological and physiological adaptations. Identification of the molecular basis of these traits can provide novel understanding for vertebrate biology and medicine. Here, we study snake biology using the genome sequence of the Burmese python (Python molurus bivittatus), a model of extreme physiological and metabolic adaptation. We compare the python and king cobra genomes along with genomic samples from other snakes and perform transcriptome analysis to gain insights into the extreme phenotypes of the python. We discovered rapid and massive transcriptional responses in multiple organ systems that occur on feeding and coordinate major changes in organ size and function. Intriguingly, the homologs of these genes in humans are associated with metabolism, development, and pathology. We also found that many snake metabolic genes have undergone positive selection, which together with the rapid evolution of mitochondrial proteins, provides evidence for extensive adaptive redesign of snake metabolic pathways. Additional evidence for molecular adaptation and gene family expansions and contractions is associated with major physiological and phenotypic adaptations in snakes; genes involved are related to cell cycle, development, lungs, eyes, heart, intestine, and skeletal structure, including GRB2-associated binding protein 1, SSH, WNT16, and bone morphogenetic protein 7. Finally, changes in repetitive DNA content, guanine-cytosine isochore structure, and nucleotide substitution rates indicate major shifts in the structure and evolution of snake genomes compared with other amniotes. Phenotypic and physiological novelty in snakes seems to be driven by system-wide coordination of protein adaptation, gene expression, and changes in the structure of the genome.

Genetic consequences of postglacial range expansion in two codistributed rodents (genus Dipodomys) depend on ecology and genetic locus

How does range expansion affect genetic diversity in species with different ecologies, and do different types of genetic markers lead to different conclusions? We addressed these questions by assessing the genetic consequences of postglacial range expansion using mitochondrial DNA (mtDNA) and nuclear restriction site‐associated DNA (RAD) sequencing in two congeneric and codistributed rodents with different ecological characteristics: the desert kangaroo rat (Dipodomys deserti), a sand specialist, and the Merriams kangaroo rat (Dipodomys merriami), a substrate generalist. For each species, we compared genetic variation between populations that retained stable distributions throughout glacial periods and those inferred to have expanded since the last glacial maximum. Our results suggest that expanded populations of both species experienced a loss of private mtDNA haplotypes and differentiation among populations, as well as a loss of nuclear single‐nucleotide polymorphism (SNP) private alleles and polymorphic loci. However, only D. deserti experienced a loss of nucleotide diversity (both mtDNA and nuclear) and nuclear heterozygosity. For all indices of diversity and differentiation that showed reduced values in the expanded areas, D. deserti populations experienced a greater degree of loss than did D. merriami populations. Additionally, patterns of loss in genetic diversity in expanded populations were substantially less extreme (by two orders of magnitude in some cases) for nuclear SNPs in both species compared to that observed for mitochondrial data. Our results demonstrate that ecological characteristics may play a role in determining genetic variation associated with range expansions, yet mtDNA diversity loss is not necessarily accompanied by a matched magnitude of loss in nuclear diversity.

EpiRADseq: scalable analysis of genomewide patterns of methylation using next‐generation sequencing

Summary Research addressing the role of epigenetics in a diversity of experimental and natural systems is rapidly accumulating. Diverse methods have been developed to study epigenetic states, including bisulphite sequencing and AFLP-based approaches. However, existing methods are sometimes difficult to apply to non-traditional model organisms that lack genomic resources (bisulphite sequencing), and can fail to be economical and readily scalable to diverse research questions because of reliance on traditional Sanger sequencing (AFLP approaches). Here we develop a reduced-representation library-based approach that is scalable and economical to quantitatively compare patterns of genomewide methylation. This approach shares substantial similarity to the now widely used double digest restriction-site associated DNA sequencing-based method (ddRADseq), except that it utilizes a methylation-sensitive restriction enzyme. This method therefore identifies changes in the genomic methylation state of cytosine (to 5-methylcytosine; 5mC) by sampling loci (via next-generation sequencing) that are not methylated within a sample. We test this method to identify shifts in the epigenome of clonal water fleas (Daphnia ambigua) in response to exposure to fish predator cues, which are known to induce transgenerational changes in life-history traits. We found evidence for differential transgenerational responses (inferred via significant shifts in the methylation state of sampled loci) to predator cues among our treatment groups and remarkably consistent responses within treatment groups. Our results demonstrate that this method is capable of producing highly repeatable results even without the use of a reference genome. Applications of this general method are broad and diverse and include the analysis of epigenetic shifts in both experimental and natural study systems.

Incipient speciation with biased gene flow between two lineages of the Western Diamondback Rattlesnake (Crotalus atrox)

We used mitochondrial DNA sequence data from 151 individuals to estimate population genetic structure across the range of the Western Diamondback Rattlesnake (Crotalus atrox), a widely distributed North American pit viper. We also tested hypotheses of population structure using double-digest restriction site associated DNA (ddRADseq) data, incorporating thousands of nuclear genome-wide SNPs from 42 individuals. We found strong mitochondrial support for a deep divergence between eastern and western C. atrox populations, and subsequent intermixing of these populations in the Inter-Pecos region of the United States and Mexico. Our nuclear RADseq data also identify these two distinct lineages of C. atrox, and provide evidence for nuclear admixture of eastern and western alleles across a broad geographic region. We identified contrasting patterns of mitochondrial and nuclear genetic variation across this genetic fusion zone that indicate partially restricted patterns of gene flow, which may be due to either pre- or post-zygotic isolating mechanisms. The failure of these two lineages to maintain complete genetic isolation, and evidence for partially-restricted gene flow, imply that these lineages were in the early stages of speciation prior to secondary contact.

The Discovery of XY Sex Chromosomes in a Boa and Python

For over 50 years, biologists have accepted that all extant snakes share the same ZW sex chromosomes derived from a common ancestor [1-3], with different species exhibiting sex chromosomes at varying stages of differentiation. Accordingly, snakes have been a well-studied model for sex chromosome evolution in animals [1, 4]. A review of the literature, however, reveals no compelling support that boas and pythons possess ZW sex chromosomes [2, 5]. Furthermore, phylogenetic patterns of facultative parthenogenesis in snakes and a sex-linked color mutation in the ball python (Python regius) are best explained by boas and pythons possessing an XY sex chromosome system [6, 7]. Here we demonstrate that a boa (Boa imperator) and python (Python bivittatus) indeed possess XY sex chromosomes, based on the discovery of male-specific genetic markers in both species. We use these markers, along with transcriptomic and genomic data, to identify distinct sex chromosomes in boas and pythons, demonstrating that XY systems evolved independently in each lineage. This discovery highlights the dynamic evolution of vertebrate sex chromosomes and further enhances the value of snakes as a model for studying sex chromosome evolution.

Phylogeographic and population genetic analyses reveal multiple species of Boa and independent origins of insular dwarfism

Boa is a Neotropical genus of snakes historically recognized as monotypic despite its expansive distribution. The distinct morphological traits and color patterns exhibited by these snakes, together with the wide diversity of ecosystems they inhabit, collectively suggest that the genus may represent multiple species. Morphological variation within Boa also includes instances of dwarfism observed in multiple offshore island populations. Despite this substantial diversity, the systematics of the genus Boa has received little attention until very recently. In this study we examined the genetic structure and phylogenetic relationships of Boa populations using mitochondrial sequences and genome-wide SNP data obtained from RADseq. We analyzed these data at multiple geographic scales using a combination of phylogenetic inference (including coalescent-based species delimitation) and population genetic analyses. We identified extensive population structure across the range of the genus Boa and multiple lines of evidence for three widely-distributed clades roughly corresponding with the three primary land masses of the Western Hemisphere. We also find both mitochondrial and nuclear support for independent origins and parallel evolution of dwarfism on offshore island clusters in Belize and Cayos Cochinos Menor, Honduras.

Genetic surfing, not allopatric divergence, explains spatial sorting of mitochondrial haplotypes in venomous coralsnakes.

Strong spatial sorting of genetic variation in contiguous populations is often explained by local adaptation or secondary contact following allopatric divergence. A third explanation, spatial sorting by stochastic effects of range expansion, has been considered less often though theoretical models suggest it should be widespread, if ephemeral. In a study designed to delimit species within a clade of venomous coralsnakes, we identified an unusual pattern within the Texas coral snake (Micrurus tener): strong spatial sorting of divergent mitochondrial (mtDNA) lineages over a portion of its range, but weak sorting of these lineages elsewhere. We tested three alternative hypotheses to explain this pattern—local adaptation, secondary contact following allopatric divergence, and range expansion. Collectively, near panmixia of nuclear DNA, the signal of range expansion associated sampling drift, expansion origins in the Gulf Coast of Mexico, and species distribution modeling suggest that the spatial sorting of divergent mtDNA lineages within M. tener has resulted from genetic surfing of standing mtDNA variation—not local adaptation or allopatric divergence. Our findings highlight the potential for the stochastic effects of recent range expansion to mislead estimations of population divergence made from mtDNA, which may be exacerbated in systems with low vagility, ancestral mtDNA polymorphism, and male‐biased dispersal.

Two low coverage bird genomes and a comparison of reference-guided versus de novo genome assemblies

As a greater number and diversity of high-quality vertebrate reference genomes become available, it is increasingly feasible to use these references to guide new draft assemblies for related species. Reference-guided assembly approaches may substantially increase the contiguity and completeness of a new genome using only low levels of genome coverage that might otherwise be insufficient for de novo genome assembly. We used low-coverage (∼3.5–5.5x) Illumina paired-end sequencing to assemble draft genomes of two bird species (the Gunnison Sage-Grouse, Centrocercus minimus, and the Clarks Nutcracker, Nucifraga columbiana). We used these data to estimate de novo genome assemblies and reference-guided assemblies, and compared the information content and completeness of these assemblies by comparing CEGMA gene set representation, repeat element content, simple sequence repeat content, and GC isochore structure among assemblies. Our results demonstrate that even lower-coverage genome sequencing projects are capable of producing informative and useful genomic resources, particularly through the use of reference-guided assemblies.

Rapid changes in gene expression direct rapid shifts in intestinal form and function in the Burmese python after feeding

Snakes provide a unique and valuable model system for studying the extremes of physiological remodeling because of the ability of some species to rapidly upregulate organ form and function upon feeding. The predominant model species used to study such extreme responses has been the Burmese python because of the extreme nature of postfeeding response in this species. We analyzed the Burmese python intestine across a time series, before, during, and after feeding to understand the patterns and timing of changes in gene expression and their relationship to changes in intestinal form and function upon feeding. Our results indicate that >2,000 genes show significant changes in expression in the small intestine following feeding, including genes involved in intestinal morphology and function (e.g., hydrolases, microvillus proteins, trafficking and transport proteins), as well as genes involved in cell division and apoptosis. Extensive changes in gene expression occur surprisingly rapidly, within the first 6 h of feeding, coincide with changes in intestinal morphology, and effectively return to prefeeding levels within 10 days. Collectively, our results provide an unprecedented portrait of parallel changes in gene expression and intestinal morphology and physiology on a scale that is extreme both in the magnitude of changes, as well as in the incredibly short time frame of these changes, with up- and downregulation of expression and function occurring in the span of 10 days. Our results also identify conserved vertebrate signaling pathways that modulate these responses, which may suggest pathways for therapeutic modulation of intestinal function in humans.

Microsatellite landscape evolutionary dynamics across 450 million years of vertebrate genome evolution

The evolutionary dynamics of simple sequence repeats (SSRs or microsatellites) across the vertebrate tree of life remain largely undocumented and poorly understood. In this study, we analyzed patterns of genomic microsatellite abundance and evolution across 71 vertebrate genomes. The highest abundances of microsatellites exist in the genomes of ray-finned fishes, squamate reptiles, and mammals, while crocodilian, turtle, and avian genomes exhibit reduced microsatellite landscapes. We used comparative methods to infer evolutionary rates of change in microsatellite abundance across vertebrates and to highlight particular lineages that have experienced unusually high or low rates of change in genomic microsatellite abundance. Overall, most variation in microsatellite content, abundance, and evolutionary rate is observed among major lineages of reptiles, yet we found that several deeply divergent clades (i.e., squamate reptiles and mammals) contained relatively similar genomic microsatellite compositions. Archosauromorph reptiles (turtles, crocodilians, and birds) exhibit reduced genomic microsatellite content and the slowest rates of microsatellite evolution, in contrast to squamate reptile genomes that have among the highest rates of microsatellite evolution. Substantial branch-specific shifts in SSR content in primates, monotremes, rodents, snakes, and fish are also evident. Collectively, our results support multiple major shifts in microsatellite genomic landscapes among vertebrates.