Genome-scale Profiling Reveals Noncoding Loci Carry Higher Proportions of Concordant Data

Many evolutionary relationships remain controversial despite whole-genome sequencing data. These controversies arise in part due to challenges associated with accurately modeling the complex phylogenetic signal coming from genomic regions experiencing distinct evolutionary forces. Here we examine how different regions of the genome support or contradict well-established hypotheses among three mammal groups using millions of orthologous parsimony-informative biallelic sites [PIBS] distributed across primate, rodent, and Pecora genomes. We compared PIBS concordance percentages among locus types (e.g. coding sequences, introns, intergenic regions), and contrasted PIBS utility over evolutionary timescales. Sites derived from noncoding sequences provided more data and proportionally more concordant sites compared with those from coding sequences [CDS] in all clades. CDS PIBS were also predominant drivers of tree incongruence in two cases of topological conflict. PIBS derived from most locus types provided surprisingly consistent support for splitting events spread across the timescales we examined, although we find evidence that CDS and intronic PIBS may, respectively and to a limited degree, inform disproportionately about older and younger splits. In this era of accessible whole genome sequence data, these results (1) suggest benefits to more intentionally focusing on noncoding loci as robust data for tree inference, and (2) reinforce the importance of accurate modeling, especially when using CDS data

Studies on the molecular underpinnings of sex determination mechanism evolution and molecular sexing tools in turtles

Sex determination mechanisms (SDMs) direct the development of individuals towards a male or female fate, and in vertebrates they are typically controlled by an individual’s genotypic content (genotypic sex determination, GSD) or through an environmental cue experienced during development, mainly temperature (temperature-dependent sex determination, TSD). Among vertebrates, SDMs are surprisingly labile, transitioning between different forms of TSD and GSD in some lineages more than others. Turtles represent a model clade to study SDM evolution, as multiple independent transitions between TSD and GSD have occurred throughout their evolution and a growing number of genomic datasets have become available. This dissertation examines the molecular underpinnings of SDM evolution in turtles while also providing tools that enable studies of sex determination across taxa and of sex-specific traits. In Chapter 2, I examine the molecular evolution of a suite of vertebrate sex determining genes in turtles, contrasting their evolutionary rates to those of other major vertebrate clades and also among turtle lineages. Furthermore, I compare the evolutionary rates of turtle lineages which have undergone SDM transitions versus those that have not. I then compare the relative evolutionary rates of turtles that have transitioned from TSD-to-GSD against lineages which have possibly transitioned from GSD-to-TSD. Finally, I discuss amino acid substitutions which occur in the functional domains of key sex determining genes along transitional branches, providing targets for future research. In Chapter 3, I present an analytical pipeline which can diagnose sex with 100% accuracy in the ZZ/ZW spiny softshell turtle Apalone spinifera by leveraging a previously described sex-biased copy number variation of rDNA clusters between the sexes. The pipeline is also applied to a previously published dataset of circulating hormonal concentrations in the snapping turtle Chelydra serpentina, where it shows greater than 85% accuracy in sex diagnosis. In Chapter 4, I lay out a bioinformatics pipeline which details the sample collection, sequencing, and analysis of sex-specific DNA libraries, which can be used to identify sex-linked DNA sequences in any taxon with sufficient genetic differentiation between the sexes. This information is leveraged to create sex-diagnostic PCR primers which are 100% accurate at diagnosing sex in the focal taxa, the ZZ/ZW A. spinifera and the XX/XY wood turtle Glyptemys insculpta. Furthermore, primers designed against the focal taxa data are also applied successfully to related species, expanding the utility of the pipeline, while simultaneously providing the first definitive evidence that the bog turtle Glyptemys muhlenbergii has an XX/XY sex chromosome system. Together these chapters provide data about the proximate mechanisms of SDM evolution in turtles, which are necessary to begin to understand the ultimate explanations for why SDM evolution is so labile across taxa

Physical Mapping and Refinement of the Painted Turtle Genome (Chrysemys picta) Inform Amniote Genome Evolution and Challenge Turtle-Bird Chromosomal Conservation

Comparative genomics continues illuminating amniote genome evolution, but for many lineages our understanding remains incomplete. Here, we refine the assembly (CPI 3.0.3 NCBI AHGY00000000.2) and develop a cytogenetic map of the painted turtle (Chrysemys picta—CPI) genome, the first in turtles and in vertebrates with temperature-dependent sex determination. A comparison of turtle genomes with those of chicken, selected nonavian reptiles, and human revealed shared and novel genomic features, such as numerous chromosomal rearrangements. The largest conserved syntenic blocks between birds and turtles exist in four macrochromosomes, whereas rearrangements were evident in these and other chromosomes, disproving that turtles and birds retain fully conserved macrochromosomes for greater than 300 Myr. C-banding revealed large heterochromatic blocks in the centromeric region of only few chromosomes. The nucleolar-organizing region (NOR) mapped to a single CPI microchromosome, whereas in some turtles and lizards the NOR maps to nonhomologous sex-chromosomes, thus revealing independent translocations of the NOR in various reptilian lineages. There was no evidence for recent chromosomal fusions as interstitial telomeric-DNA was absent. Some repeat elements (CR1-like, Gypsy) were enriched in the centromeres of five chromosomes, whereas others were widespread in the CPI genome. Bacterial artificial chromosome (BAC) clones were hybridized to 18 of the 25 CPI chromosomes and anchored to a G-banded ideogram. Several CPI sex-determining genes mapped to five chromosomes, and homology was detected between yet other CPI autosomes and the globally nonhomologous sex chromosomes of chicken, other turtles, and squamates, underscoring the independent evolution of vertebrate sex-determining mechanisms

Screening the PRISM Library against Staphylococcus aureus Reveals a Sesquiterpene Lactone from Liriodendron tulipifera with Inhibitory Activity

Infections caused by the bacterium Staphylococcus aureus continue to pose threats to human health and put a financial burden on the healthcare system. The overuse of antibiotics has contributed to mutations leading to the emergence of methicillin-resistant S. aureus, and there is a critical need for the discovery and development of new antibiotics to evade drug-resistant bacteria. Medicinal plants have shown promise as sources of new small-molecule therapeutics with potential uses against pathogenic infections. The principal Rhode Island secondary metabolite (PRISM) library is a botanical extract library generated from specimens in the URI Youngken Medicinal Garden by upper-division undergraduate students. PRISM extracts were screened for activity against strains of methicillin-susceptible S. aureus (MSSA). An extract generated from the tulip tree (Liriodendron tulipifera) demonstrated growth inhibition against MSSA, and a bioassay-guided approach identified a sesquiterpene lactone, laurenobiolide, as the active constituent. Intriguingly, its isomers, tulipinolide and epi-tulipinolide, lacked potent activity against MSSA. Laurenobiolide also proved to be more potent against MSSA than the structurally similar sesquiterpene lactones, costunolide and dehydrocostus lactone. Laurenobiolide was the most abundant in the twig bark of the tulip tree, supporting the twig bark’s historical and cultural usage in poultices and teas

The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage

Background: We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing.Results: Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented.Conclusions: Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders

Studies on the molecular underpinnings of sex determination mechanism evolution and molecular sexing tools in turtles

Sex determination mechanisms (SDMs) direct the development of individuals towards a male or female fate, and in vertebrates they are typically controlled by an individual’s genotypic content (genotypic sex determination, GSD) or through an environmental cue experienced during development, mainly temperature (temperature-dependent sex determination, TSD). Among vertebrates, SDMs are surprisingly labile, transitioning between different forms of TSD and GSD in some lineages more than others. Turtles represent a model clade to study SDM evolution, as multiple independent transitions between TSD and GSD have occurred throughout their evolution and a growing number of genomic datasets have become available. This dissertation examines the molecular underpinnings of SDM evolution in turtles while also providing tools that enable studies of sex determination across taxa and of sex-specific traits. In Chapter 2, I examine the molecular evolution of a suite of vertebrate sex determining genes in turtles, contrasting their evolutionary rates to those of other major vertebrate clades and also among turtle lineages. Furthermore, I compare the evolutionary rates of turtle lineages which have undergone SDM transitions versus those that have not. I then compare the relative evolutionary rates of turtles that have transitioned from TSD-to-GSD against lineages which have possibly transitioned from GSD-to-TSD. Finally, I discuss amino acid substitutions which occur in the functional domains of key sex determining genes along transitional branches, providing targets for future research. In Chapter 3, I present an analytical pipeline which can diagnose sex with 100% accuracy in the ZZ/ZW spiny softshell turtle Apalone spinifera by leveraging a previously described sex-biased copy number variation of rDNA clusters between the sexes. The pipeline is also applied to a previously published dataset of circulating hormonal concentrations in the snapping turtle Chelydra serpentina, where it shows greater than 85% accuracy in sex diagnosis. In Chapter 4, I lay out a bioinformatics pipeline which details the sample collection, sequencing, and analysis of sex-specific DNA libraries, which can be used to identify sex-linked DNA sequences in any taxon with sufficient genetic differentiation between the sexes. This information is leveraged to create sex-diagnostic PCR primers which are 100% accurate at diagnosing sex in the focal taxa, the ZZ/ZW A. spinifera and the XX/XY wood turtle Glyptemys insculpta. Furthermore, primers designed against the focal taxa data are also applied successfully to related species, expanding the utility of the pipeline, while simultaneously providing the first definitive evidence that the bog turtle Glyptemys muhlenbergii has an XX/XY sex chromosome system. Together these chapters provide data about the proximate mechanisms of SDM evolution in turtles, which are necessary to begin to understand the ultimate explanations for why SDM evolution is so labile across taxa.</p

Utilizing Big Data to Identify Tiny Toxic Components: Digitalis

The botanical genus Digitalis is equal parts colorful, toxic, and medicinal, and its bioactive compounds have a long history of therapeutic use. However, with an extremely narrow therapeutic range, even trace amounts of Digitalis can cause adverse effects. Using chemical methods, the United States Food and Drug Administration traced a 1997 case of Digitalis toxicity to a shipment of Plantago (a common ingredient in dietary supplements marketed to improve digestion) contaminated with Digitalis lanata. With increased accessibility to next generation sequencing technology, here we ask whether this case could have been cracked rapidly using shallow genome sequencing strategies (e.g., genome skims). Using a modified implementation of the Site Identification from Short Read Sequences (SISRS) bioinformatics pipeline with whole-genome sequence data, we generated over 2 M genus-level single nucleotide polymorphisms in addition to species-informative single nucleotide polymorphisms. We simulated dietary supplement contamination by spiking low quantities (0–10%) of Digitalis whole-genome sequence data into a background of commonly used ingredients in products marketed for “digestive cleansing” and reliably detected Digitalis at the genus level while also discriminating between Digitalis species. This work serves as a roadmap for the development of novel DNA-based assays to quickly and reliably detect the presence of toxic species such as Digitalis in food products or dietary supplements using genomic methods and highlights the power of harnessing the entire genome to identify botanical species

MeDIP-seq and nCpG analyses illuminate sexually dimorphic methylation of gonadal development genes with high historic methylation in turtle hatchlings with temperature-dependent sex determination

Background: DNA methylation alters gene expression but not DNA sequence and mediates some cases of phenotypic plasticity. Temperature-dependent sex determination (TSD) epitomizes phenotypic plasticity where environmental temperature drives embryonic sexual fate, as occurs commonly in turtles. Importantly, the temperature-specific transcription of two genes underlying gonadal differentiation is known to be induced by differential methylation in TSD fish, turtle and alligator. Yet, how extensive is the link between DNA methylation and TSD remains unclear. Here we test for broad differences in genome-wide DNA methylation between male and female hatchling gonads of the TSD painted turtle Chrysemys picta using methyl DNA immunoprecipitation sequencing, to identify differentially methylated candidates for future study. We also examine the genome-wide nCpG distribution (which affects DNA methylation) in painted turtles and test for historic methylation in genes regulating vertebrate gonadogenesis. Results: Turtle global methylation was consistent with other vertebrates (57% of the genome, 78% of all CpG dinucleotides). Numerous genes predicted to regulate turtle gonadogenesis exhibited sex-specific methylation and were proximal to methylated repeats. nCpG distribution predicted actual turtle DNA methylation and was bimodal in gene promoters (as other vertebrates) and introns (unlike other vertebrates). Differentially methylated genes, including regulators of sexual development, had lower nCpG content indicative of higher historic methylation. Conclusions: Ours is the first evidence suggesting that sexually dimorphic DNA methylation is pervasive in turtle gonads (perhaps mediated by repeat methylation) and that it targets numerous regulators of gonadal development, consistent with the hypothesis that it may regulate thermosensitive transcription in TSD vertebrates. However, further research during embryogenesis will help test this hypothesis and the alternative that instead, most differential methylation observed in hatchlings is the by-product of sexual differentiation and not its cause.This article is published as Radhakrishnan, Srihari, Robert Literman, Beatriz Mizoguchi, and Nicole Valenzuela. "MeDIP-seq and nCpG analyses illuminate sexually dimorphic methylation of gonadal development genes with high historic methylation in turtle hatchlings with temperature-dependent sex determination." Epigenetics & chromatin 10 (2017): 28. doi: 10.1186/s13072-017-0136-2.</p

MOESM1 of MeDIP-seq and nCpG analyses illuminate sexually dimorphic methylation of gonadal development genes with high historic methylation in turtle hatchlings with temperature-dependent sex determination

Additional file 1. Table S1: Genes with distinct hypermethylated sex-specific windows within the same gene. Table S2: Categories enriched (p = 0.05) in hypermethylated genes at male-producing temperature (26 °C). Table S3: Categories enriched (p = 0.05) in hypermethylated genes at female-producing temperature (31 °C). Tables S4 through S11—differentially methylated genes in C. picta hatchling methylomes along with RPKM, fold change and edgeR p value: Table S4: Heat shock genes. Table S5: Androgen-/estrogen-related genes. Table S6: Kinases. Table S7: Histone-related genes. Table S8: Ubiquitin-related genes. Table S9: Transient receptor potential genes. Table S10: Genes involved in cell proliferation. Table S11: Germline-related genes. Table S12: Number of differentially methylated genes by GO term present in testis and ovaries of C. picta hatchlings. Table S13: Comprehensive list of differentially methylated genes and associated GO pathways between testis and ovaries of C. picta hatchlings. Table S14: Statistics of all differentially methylated genes between testis and ovaries of C. picta hatchlings. Table S15: Genes differentially expressed in the C. picta transcriptome (stage 22) [42] and differentially methylated in the hatchling methylome (this study). Table S16: Relative abundance distribution of repeat categories as a fraction of the genome. Table S17: Overlapping genes between C. picta hatchling methylomes and the methylomes of tongue sole [18]

Transcriptomic responses to environmental temperature by turtles with temperature-dependent and genotypic sex determination assessed by RNAseq inform the genetic architecture of embryonic gonadal development

Vertebrate sexual fate is decided primarily by the individual’s genotype (GSD), by the environmental temperature during development (TSD), or both. Turtles exhibit TSD and GSD, making them ideal to study the evolution of sex determination. Here we analyze temperature-specific gonadal transcriptomes (RNA-sequencing validated by qPCR) of painted turtles (Chrysemys picta TSD) before and during the thermosensitive period, and at equivalent stages in soft-shell turtles (Apalone spinifera—GSD), to test whether TSD’s and GSD’s transcriptional circuitry is identical but deployed differently between mechanisms. Our data show that most elements of the mammalian urogenital network are active during turtle gonadogenesis, but their transcription is generally more thermoresponsive in TSD than GSD, and concordant with their sex-specific function in mammals [e.g., upregulation of Amh, Ar, Esr1, Fog2, Gata4, Igf1r, Insr, and Lhx9 at male-producing temperature, and of β-catenin, Foxl2, Aromatase (Cyp19a1), Fst, Nf-kb, Crabp2 at female-producing temperature in Chrysemys]. Notably, antagonistic elements in gonadogenesis (e.g., β-catenin and Insr) were thermosensitive only in TSD early-embryos. Cirbp showed warm-temperature upregulation in both turtles disputing its purported key TSD role. Genes that may convert thermal inputs into sex-specific development (e.g., signaling and hormonal pathways, RNA-binding and heat-shock) were differentially regulated. Jak-Stat, Nf-κB, retinoic-acid, Wnt, and Mapk-signaling (not Akt and Ras-signaling) potentially mediate TSD thermosensitivity. Numerous species-specific ncRNAs (including Xist) were differentially-expressed, mostly upregulated at colder temperatures, as were unannotated loci that constitute novel TSD candidates. Cirbp showed warm-temperature upregulation in both turtles. Consistent transcription between turtles and alligator revealed putatively-critical reptilian TSD elements for male (Sf1, Amh, Amhr2) and female (Crabp2 and Hspb1) gonadogenesis. In conclusion, while preliminary, our data helps illuminate the regulation and evolution of vertebrate sex determination, and contribute genomic resources to guide further research into this fundamental biological process.This article is published as Radhakrishnan, Srihari, Robert Literman, Jennifer Neuwald, Andrew Severin, and Nicole Valenzuela. "Transcriptomic responses to environmental temperature by turtles with temperature-dependent and genotypic sex determination assessed by RNAseq inform the genetic architecture of embryonic gonadal development." PloS one 12, no. 3 (2017): e0172044. doi: 10.1371/journal.pone.0172044. Posted with permission.</p