The red-eared slider, Trachemys scripta elegans (Wied, 1938), has been introduced worldwide, partly because of the exotic pet trade in the 1980s and 1990s. When T. s. elegans is released or escapes into natural environments, it often establishes new feral populations due to its tolerance for a variety of aquatic ecosystems. Therefore, it is now considered one of the most invasive species in the world because it can compete with native turtle species. In the present study, our objectives were to identify the potential for polystome spillover and spillback resulting from the introduction of the red-eared slider into new environments in North America. Fieldwork investigations were thus conducted mainly in aquatic habitats in Florida and North Carolina, United States, but also in Connecticut, Indiana, Kansas, Maine, Nebraska and New York. Using DNA barcoding based on cytochrome c oxidase I (COI) sequences, we surveyed the species diversity of polystome within American freshwater turtles. These included T. s. elegans but also Apalone ferox, Apalone spinifera, Chelydra serpentina, Chrysemys picta, Kinosternon baurii, Pseudemys spp., Sternotherus minor and Sternotherus odoratus. Genetic evidence confirmed that invasive populations of T. s. elegans in southern Europe have transmitted their own polystomes to native host species following spillover effects, and revealed here that T. s. elegans in non-indigenous habitats in the United States acts as a new reservoir of infection for native polystomes following spillback effects, thus increasing indigenous parasite transmission in the wild. Together, these findings raise further concern about the spread of non-native turtles and their impact on parasite transmission.

Frogs are an ecologically diverse and phylogenetically ancient group of anuran amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus . Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis , along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui , Engystomops pustulosus , and Hymenochirus boettgeri . Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., arm-preserving) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding surrounded by pericentromeric LINE/L1 elements. This work explores the structure of chromosomes across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (Hi-C) data for all species. Abundant satellite repeats occupy the unusually long (~20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible associations of centromeric chromatin and of telomeres, reflecting a Rabl-like configuration. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.

Biological diversity sustains humanity. It is the nature of the evolutionary process for biodiversity to wax and wane over time, but biodiversity is currently declining at rates undocumented in human history and anthropogenic activities are the primary drivers of biodiversity losses. A variety of solutions have been proposed as means to protect nature and, more specifically, biodiversity. The book you hold posits an idea that may work: the commodification of nature through the lens of biomedical benefits. It remains to be proven whether our decisions and actions will follow this approach and, more so, whether we can pursue this approach in a sustainable relationship with nature, curbing the ongoing rapid species loss while maximizing medical benefits. The challenges are many, cross-cultural, and span engineering and scientific disciplines. The entire value chain iterating between biodiversity and biomedicine requires significant investments of time, money, and expertise if this endeavor is to work. Briefly, challenges include: (1) discovering and documenting species diversity; (2) cataloging traditional medicines developed from these species, including medicinal preparations (e.g., fermentation, desiccation, etc.); (3) assaying genetic and chemical diversity of natural products; (4) testing promising metabolites and other compounds for medical applicability and efficacy (and in turn developing drugs as appropriate); (5) determining why some conservation efforts succeed and others fail; (6) negotiating policies that incentivize and empower local communities to conserve the diversity in their vicinity, while also enacting policies that protect ecosystems from biopiracy and illicit harvesting and trade; and (7) strengthening and developing policy and institutional frameworks to codify and maintain working solutions. Sustainable solutions that bridge biodiversity to biomedicine may include the adoption of policies that shift paradigms around "ownership" of nature and the ecosystem services humans enjoy from diverse systems. Here we provide a context for the concept of commodifying ecosystem services as natural resources.

With about 25% of life-saving pharmaceutical drugs derived from plants, unexplored flora offer much promise as a source of new pharmacologically active molecules. Despite the number of challenges associated with plants as a drug discovery source, the thousands to millions of different molecules that plants produce cannot be disregarded in the wake of drug resistance and emerging diseases. This book chapter thus provides an insight of the historical evidence of plants as a source of drugs, with a view to discussing the barriers that limit further research and development in this area, and provides an overview of how these challenges could be overcome. The importance of partnerships among scientists, pharmaceutical industries, and indigenous communities to identify species and isolate compounds, social scientists to assess community needs and ensure community empowerment, and policy makers to produce legislation to guide protection and to develop models for sustainable agriculture and research involving modern biotechnology and chemistry are proposed.

The skin secretions of many frogs have genetically-encoded, endogenous antimicrobial peptides (AMPs). Other species, especially aposematic poison frogs, secrete exogenously derived alkaloids that serve as potent defense molecules. The origins of these defense systems are not clear, but a novel bile-acid derived metabolite, tauromantellic acid, was recently discovered and shown to be endogenous in poison frogs (Mantella, Dendrobates, and Epipedobates). These observations raise questions about the evolutionary history of AMP genetic elements, the mechanism and function of tauromatellic acid production, and links between these systems. To understand the diversity and expression of AMPs among frogs, we assembled skin transcriptomes of 13 species across the anuran phylogeny. Our analyses revealed a diversity of AMPs and AMP expression levels across the phylogenetic history of frogs, but no observations of AMPs in Mantella. We examined genes expressed in the bile-acid metabolic pathway and found that CYP7A1 (Cytochrome P450), BAAT (bile acid-CoA: amino acid N-acyltransferase), and AMACR (alpha-methylacyl-CoA racemase) were highly expressed in the skin of M. betsileo and either lowly expressed or absent in other frog species. In particular, CYP7A1 catalyzes the first reaction in the cholesterol catabolic pathway and is the rate-limiting step in regulation of bile acid synthesis, suggesting unique activation of the bile acid pathway in Mantella skin. The activation of the bile acid pathway in the skin of Mantella and the lack of observed AMPs fuel new questions about the evolution of defense compounds and the ectopic expression of the bile-acid pathway.

Unisexual Ambystoma are difficult to distinguish from the sexual salamanders with which they interact, and all of these species are of conservation concern throughout much of their range. We examined egg masses to develop an efficient, affordable method to differentiate unisexual salamanders and Jefferson Salamanders (A. jeffersonianum). We developed a technique based on photographs of egg masses from three populations held in the laboratory and tested it in the field. Using photographs, we first determined the long axis of the egg mass, and then drew a line perpendicular to the long axis and counted the maximum number of embryos or egg membranes that intersected with this line. The resulting Embryo Density Index was higher in egg masses of Jefferson Salamander (mean = 6.3 +/- 2.0 SD) than in unisexuals (mean = 3.8 +/- 1.3). Likewise, the Egg Membrane Density Index was higher in egg masses of Jefferson Salamanders (mean = 7.7 +/- 2.3) than in unisexuals (mean = 4.6 +/- 1.6). Although those results were statistically significant, there was considerable overlap in egg density index values between salamander biotypes, making the methods more appropriate for population-level analyses than for determining species identity of a given individual egg mass. Our scale-independent method of visually assessing the spacing of eggs within a mass can be used across developmental stages, and it enables rapid data collection with very little equipment or cost.

BACKGROUND:Animals with polyploid, hybrid nuclei offer a challenge for models of gene expression and regulation during embryogenesis. To understand how such organisms proceed through development, we examined the timing and prevalence of mortality among embryos of unisexual salamanders in the genus Ambystoma.RESULTS:Our regional field surveys suggested that heightened rates of embryo mortality among unisexual salamanders begin in the earliest stages of embryogenesis. Although we expected elevated mortality after zygotic genome activation in the blastula stage, this is not what we found among embryos which we reared in the laboratory. Once embryos entered the first cleavage stage, we found no difference in mortality rates between unisexual salamanders and their bisexual hosts. Our results are consistent with previous studies showing high rates of unisexual mortality, but counter to reports that heightened embryo mortality continues throughout embryo development.CONCLUSIONS:Possible causes of embryonic mortality in early embryogenesis suggested by our results include abnormal maternal loading of RNA during meiosis and barriers to insemination. The surprising survival rates of embryos post-cleavage invites further study of how genes are regulated during development in such polyploid hybrid organisms.

Heteromorphic sex chromosomes have evolved repeatedly among vertebrate lineages despite largely deleterious reductions in gene dose. Understanding how this gene dose problem is overcome is hampered by the lack of genomic information at the base of tetrapods and comparisons across the evolutionary history of vertebrates. To address this problem, we produced a chromosome-level genome assembly for the African Bullfrog ( Pyxicephalus adspersus )—an amphibian with heteromorphic ZW sex chromosomes—and discovered that the Bullfrog Z is surprisingly homologous to substantial portions of the human X. Using this new reference genome, we identified ancestral synteny among the sex chromosomes of major vertebrate lineages, showing that non-mammalian sex chromosomes are strongly associated with a single vertebrate ancestral chromosome, while mammals are associated with another that displays increased haploinsufficiency. The sex chromosomes of the African Bullfrog however, share genomic blocks with both humans and non-mammalian vertebrates, connecting the two ancestral chromosome sequences that repeatedly characterize vertebrate sex chromosomes. Our results highlight the consistency of sex-linked sequences despite sex determination system lability and reveal the repeated use of two major genomic sequence blocks during vertebrate sex chromosome evolution.

Amphibians inhabiting montane riparian zones in the Neotropics are particularly vulnerable to decline, but the reasons are poorly understood. Because environmental contaminants, endocrine disruption, and pathogens often figure prominently in amphibian declines it is imperative that we understand how these factors are potentially interrelated to affect montane populations. One possibility is that increased precipitation associated with global warming promotes the deposition of contaminants in montane regions. Increased exposure to contaminants, in turn, potentially elicits chronic elevations in circulating stress hormones that could contribute to montane population declines by compromising resistance to pathogens and/or production of sex steroids regulating reproduction. Here, we test this hypothesis by examining contaminant levels, stress and sex steroid levels, and nematode abundances in male drab treefrogs, Smilisca sordida, from lowland and montane populations in Costa Rica. We found no evidence that montane populations were more likely to possess contaminants (i.e., organochlorine, organophosphate and carbamate pesticides or benzidine and chlorophenoxy herbicides) than lowland populations. We also found no evidence of elevational differences in circulating levels of the stress hormone corticosterone, estradiol or progesterone. However, montane populations possessed lower androgen levels, hosted more nematode species, and had higher nematode abundances than lowland populations. Although these results suggested that nematodes contributed to lower androgens in montane populations, we were unable to detect a significant inverse relationship between nematode abundance and androgen level. Our results suggest that montane populations of this species are not at greater risk of exposure to contaminants or chronic stress, but implicate nematodes and compromised sex steroid levels as potential threats to montane populations.

Looking to nature for medicine is nothing new -we have been doing it for tens of thousands of years and although modern pharmaceutical science has come a long way from those ancient roots, nature is and will always be an important source of useful compounds and inspiration.Dismissing nature in this regard is a huge mistake as evolution is the greatest problem solver and the myriad compounds produced by the immense variety of species we share the planet with have been honed by three billion years of trial and error.However, with every bit of habitat that disappears under the plough or concrete we impoverish nature and deprive ourselves of potential medicines.The preservation of biodiversity is perhaps the single most important building block for achieving the 17 Sustainable Development Goals set by the United Nations.For many of these goals, the importance of Biodiversity, drug discovery, and the future of global health

Ecologists recognize the balance of nature as a complex system that arises from interacting and connected parts; disturbing the balance can affect other species in nonlinear and dynamic ways. Like ecosystems, the cell and genome are extensively connected by an abundance of molecular interactions. Upsetting the molecular balance, through changes in gene regulation, abnormal copy number variants, and changes in cell size can create consequences for genome and organismal function (1, 2). In PNAS, Gibbons et al. (3) advance the idea of genome balance further by identifying a new source of genetic variation that results in balancing transcriptional subunits of the ribosome. Using data from human and mouse, the authors characterized copy number variation (CNV) in tandemly repeated ribosomal DNA (rDNA) arrays and found that subunits of the array vary together in what they define as concerted copy number variation (cCNV). cCNV raises new questions about how CNV can function to restore balance in eukaryotic genomes, especially on the assembly and function of the ribosome. The eukaryotic ribosome is a macromolecular complex composed of about 80 single copy ribosomal proteins and 4 noncoding ribosomal RNAs whose genes are responsible for translating mRNAs into proteins in all living cells. The genes that encode these RNAs are extensively duplicated and scattered throughout the genome (4, 5). The noncoding RNA loci are especially intricate. The 45S rDNA locus is transcribed by RNA polymerase I and spliced to produce the 18S, 5.8S, and 28S rRNAs. The 5S rDNA locus is transcribed by polymerase III and produces 5S rRNA. These independently transcribed noncoding RNA units must act together to form the ribosome (Fig. 1A). The mammalian ribosome is composed of a large subunit (60S) and a small subunit (40S) that are composed of a diverse set of proteins and transcriptional arrays of …

Dosage compensation is a mechanism that facilitates changes in expression relative to DNA abundance. Intensive study of worms, flies, and human sex chromosomes during the last several decades has shown that intrinsic effects, non coding RNA, macromolecular complexes, or a combination act to manipulate expression. The strength of studying these model organisms comes from detailed genetic knowledge and an array of molecular tools, however with the advent of whole genome sequencing, it becomes possible to understand dosage compensation in a wider evolutionary context. In this chapter, I provide an overview of dosage compensation and what is known about dosage compensation in frogs and toads. Frogs and toads represent the basal tetra pods and are useful models for understanding gene dosage during early development, a window that is difficult to study in vertebrates. While few studies exist, there are curious examples of dosage compensation in frogs. Applying genomic tools in frogs and toads will provide a better understanding for the role of compensation in solving problems of abnormal gene dosage, and create new opportunities for understanding the role of dosage compensation in evolution, genome function, and early development.

A new study provides evidence that gene transposition from sex chromosomes to autosomes is a conserved phenomenon across mammalian species that rescues dosage-sensitive genes.

Young species complexes that are widespread across ecologically disparate regions offer important insights into the process of speciation because of their relevance to how local adaptation and gene flow influence diversification. We used mitochondrial DNA and up to 28 152 genomewide single nucleotide polymorphisms from polytypic barking frogs ( C raugastor augusti complex) to infer phylogenetic relationships and test for the signature of introgressive hybridization among diverging lineages. Our phylogenetic reconstructions suggest (i) a rapid P liocene– P leistocene radiation that produced at least nine distinct lineages and (ii) that geographic features of the arid C entral M exican P lateau contributed to two independent northward expansions. Despite clear lineage differentiation (many private alleles and high between‐lineage F ST scores), D ‐statistic tests, which differentiate introgression from ancestral polymorphism, allowed us to identify two putative instances of reticulate gene flow. Partitioned D ‐statistics provided evidence that these events occurred in the same direction between clades but at different points in time. After correcting for geographic distance, we found that lineages involved in hybrid gene flow interactions had higher levels of genetic variation than independently evolving lineages. These findings suggest that the nature of hybrid compatibility can be conserved overlong periods of evolutionary time and that hybridization between diverging lineages may contribute to standing levels of genetic variation.

Accurate gene model annotation of reference genomes is critical for making them useful. The modENCODE project has improved the D. melanogaster genome annotation by using deep and diverse high-throughput data. Since transcriptional activity that has been evolutionarily conserved is likely to have an advantageous function, we have performed large-scale interspecific comparisons to increase confidence in predicted annotations. To support comparative genomics, we filled in divergence gaps in the Drosophila phylogeny by generating draft genomes for eight new species. For comparative transcriptome analysis, we generated mRNA expression profiles on 81 samples from multiple tissues and developmental stages of 15 Drosophila species, and we performed cap analysis of gene expression in D. melanogaster and D. pseudoobscura. We also describe conservation of four distinct core promoter structures composed of combinations of elements at three positions. Overall, each type of genomic feature shows a characteristic divergence rate relative to neutral models, highlighting the value of multispecies alignment in annotating a target genome that should prove useful in the annotation of other high priority genomes, especially human and other mammalian genomes that are rich in noncoding sequences. We report that the vast majority of elements in the annotation are evolutionarily conserved, indicating that the annotation will be an important springboard for functional genetic testing by the Drosophila community.

Biotic and abiotic factors have been proposed to explain patterns of reproductive character displacement, but which factor is most important to character displacement of acoustic signals is not clear. Male vocalizations of the frog Pseudacris feriarum are known to undergo reproductive character displacement in areas of sympatry with P. brimleyi and P. nigrita. Despite evidence for reinforcement as an important mechanism, local adaptation via sensory drive might explain this pattern because Pseudacris breed in different habitat types and mating signals are exposed to a variety of environments. We tested the sensory drive hypothesis by playing synthesized vocalizations representing the spectrum of variation in P. feriarum at 12 different study sites. If sensory drive has occurred, then vocalizations should transmit better in the site of origin or at ecologically similar sites. We found that variation in acoustic signals did not produce better transmission in particular sites, the effect of site was uniform, and acoustic signals often transmitted better in habitats external to their origin. Ecological variation among habitats did not explain signal degradation. Our playback experiments, ecological analyses, and comparisons of different habitat types provide no support for sensory drive as a process promoting reproductive character displacement in this system. Reinforcement is the more likely primary mechanism.

Frog sex chromosomes offer an ideal system for advancing our understanding of genome evolution and function because of the variety of sex determination systems in the group, the diversity of sex chromosome maturation states, the ease of experimental manipulation during early development.After briefly reviewing sex chromosome biology generally, we focus on what is known about frog sex determination, sex chromosome evolution, and recent, genomics-facilitated advances in the field.In closing we highlight gaps in our current knowledge of frog sex chromosomes, and suggest priorities for future research that can advance broad knowledge of gene dose and sex chromosome evolution.

Modern biomedical research demands that investigators become familiar with deep sequencing data analysis, yet the vast nature of deep sequencing data creates a variety of roadblocks for biologists not familiar with the analysis of such large datasets. In this chapter, we provide an introduction to data analysis for biologists, review first principles, point out areas of concern, and suggest software tools that are becoming standards for analysis of deep sequencing data. Perhaps the biggest challenge in the analysis of deep sequencing data will be data management and storage and repeating complex, multitier computational analyses. The future of deep sequencing data analysis will be likely data-driven and rely on principles gleaned from “big data” analysis.

Background: The production of multiple transcript isoforms from one gene is a major source of transcriptome complexity. RNA-Seq experiments, in which transcripts are converted to cDNA and sequenced, allow the resolution and quantification of alternative transcript isoforms. However, methods to analyze splicing are underdeveloped and errors resulting in incorrect splicing calls occur in every experiment.Results: We used RNA-Seq data to develop sequencing and aligner error models. By applying these error models to known input from simulations, we found that errors result from false alignment to minor splice motifs and antisense stands, shifted junction positions, paralog joining, and repeat induced gaps. By using a series of quantitative and qualitative filters, we eliminated diagnosed errors in the simulation, and applied this to RNA-Seq data from Drosophila melanogaster heads. We used high-confidence junction detections to specifically interrogate local splicing differences between transcripts. This method out-performed commonly used RNA-seq methods to identify known alternative splicing events in the Drosophila sex determination pathway. We describe a flexible software package to perform these tasks called Splicing Analysis Kit (Spanki), available at http://www.cbcb umd.edu/software/spanki.Conclusions: Splice-junction centric analysis of RNA-Seq data provides advantages in specificity for detection of alternative splicing. Our software provides tools to better understand error profiles in RNA-Seq data and improve inference from this new technology. The splice-junction centric approach that this software enables will provide more accurate estimates of differentially regulated splicing than current tools.

DNA sequences on X chromosomes often have a faster rate of evolution when compared to similar loci on the autosomes, and well articulated models provide reasons why the X-linked mode of inheritance may be responsible for the faster evolution of X-linked genes. We analyzed microarray and RNA-seq data collected from females and males of six Drosophila species and found that the expression levels of X-linked genes also diverge faster than autosomal gene expression, similar to the "faster-X" effect often observed in DNA sequence evolution. Faster-X evolution of gene expression was recently described in mammals, but it was limited to the evolutionary lineages shortly following the creation of the therian X chromosome. In contrast, we detect a faster-X effect along both deep lineages and those on the tips of the Drosophila phylogeny. In Drosophila males, the dosage compensation complex (DCC) binds the X chromosome, creating a unique chromatin environment that promotes the hyper-expression of X-linked genes. We find that DCC binding, chromatin environment, and breadth of expression are all predictive of the rate of gene expression evolution. In addition, estimates of the intraspecific genetic polymorphism underlying gene expression variation suggest that X-linked expression levels are not under relaxed selective constraints. We therefore hypothesize that the faster-X evolution of gene expression is the result of the adaptive fixation of beneficial mutations at X-linked loci that change expression level in cis. This adaptive faster-X evolution of gene expression is limited to genes that are narrowly expressed in a single tissue, suggesting that relaxed pleiotropic constraints permit a faster response to selection. Finally, we present a conceptional framework to explain faster-X expression evolution, and we use this framework to examine differences in the faster-X effect between Drosophila and mammals.