Identifying hybridization and admixture using SNPs:Application of the DArTseq platforminphylogeographic research on vertebrates

Next-generation sequencing (NGS) approaches are increasingly being used to generate multi-locus data for phylogeographic and evolutionary genetics research. We detail the applicability of a restriction enzyme-mediated genome complexity reduction approach with subsequent NGS (DArTseq) in vertebrate study systems at different evolutionary and geographical scales. We present two case studies using SNP data from the DArTseq molecular marker platform. First, we used DArTseq in a large phylogeographic study of the agamid lizard Ctenophorus caudicinctus, including 91 individuals and spanning the geographical range of this species across arid Australia. A low-density DArTseq assay resulted in 28 960 SNPs, with low density referring to a comparably reduced set of identified and sequenced markers as a cost-effective approach. Second, we applied this approach to an evolutionary genetics study of a classic frog hybrid zone (Litoria ewingii–Litoria paraewingi) across 93 individuals, which resulted in 48 117 and 67 060 SNPs for a low- and high-density assay, respectively. We provide a docker-based workflow to facilitate data preparation and analysis, then analyse SNP data using multiple methods including Bayesian model-based clustering and conditional likelihood approaches. Based on comparison of results from the DArTseq platform and traditional molecular approaches, we conclude that DArTseq can be used successfully in vertebrates and will be of particular interest to researchers working at the interface between population genetics and phylogenetics, exploring species boundaries, gene exchange and hybridization. © 2017 The Authors

Differing views - can chimpanzees do level 2 perspective-taking?

We gratefully acknowledge financial support by the German National Academic Foundation.Although chimpanzees understand what others may see, it is unclear if they understand how others see things (Level 2 perspective-taking). We investigated whether chimpanzees can predict the behavior of a conspecific which is holding a mistaken perspective that differs from their own. The subject competed with a conspecific over two food sticks. While the subject could see that both were the same size, to the competitor one appeared bigger than the other. In a previously established game, the competitor chose one stick in private first and the subject chose thereafter, without knowing which of the sticks was gone. Chimpanzees and 6-year-old children chose the ‘riskier’ stick (that looked bigger to the competitor) significantly less in the game than in a nonsocial control. Children chose randomly in the control, thus showing Level 2 perspective-taking skills; in contrast, chimpanzees had a preference for the ‘riskier’ stick here, rendering it possible that they attributed their own preference to the competitor to predict her choice. We thus run a follow-up in which chimpanzees did not have a preference in the control. Now they also chose randomly in the game. We conclude that chimpanzees solved the task by attributing their own preference to the other, while children truly understood the other’s mistaken perspective.Publisher PDFPeer reviewe

Expression of P. falciparum var Genes Involves Exchange of the Histone Variant H2A.Z at the Promoter

Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes

Analysis of shared common genetic risk between amyotrophic lateral sclerosis and epilepsy

Because hyper-excitability has been shown to be a shared pathophysiological mechanism, we used the latest and largest genome-wide studies in amyotrophic lateral sclerosis (n = 36,052) and epilepsy (n = 38,349) to determine genetic overlap between these conditions. First, we showed no significant genetic correlation, also when binned on minor allele frequency. Second, we confirmed the absence of polygenic overlap using genomic risk score analysis. Finally, we did not identify pleiotropic variants in meta-analyses of the 2 diseases. Our findings indicate that amyotrophic lateral sclerosis and epilepsy do not share common genetic risk, showing that hyper-excitability in both disorders has distinct origins

Transcription of Pitx1 in Ctenophorus pictus (CP) and Pogona vitticeps (PV) embryonic limbs from Expression of a hindlimb-determining factor <i>Pitx1</i> in the forelimb of the lizard <i>Pogona vitticeps</i> during morphogenesis

With over 9000 species, squamates, which include lizards and snakes, are the largest group of reptiles and second-largest order of vertebrates, spanning a vast array of appendicular skeletal morphology. As such, they provide a promising system for examining developmental and molecular processes underlying limb morphology. Using the Central Bearded Dragon (<i>Pogona vitticeps</i>) as the primary study model, we examined limb morphometry throughout embryonic development and characterized the expression of three known developmental genes (<i>GHR, Pitx1</i> and <i>Shh</i>) from early embryonic stage through to hatchling stage via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). In this study, all genes were found to be transcribed in both the forelimb and hindlimbs of <i>P. vitticeps.</i> While the highest level of <i>GHR</i> expression occurred at the hatchling stage, <i>Pitx1</i> and <i>Shh</i> expression was greatest earlier during embryogenesis, which coincides with the onset of the differentiation between forelimb and hindlimb length. We compared our finding of <i>Pitx1</i> expression—a hindlimb-determining gene—in the forelimbs of <i>P. vitticeps</i> to that in a closely related Australian agamid lizard, <i>Ctenophorus pictus</i>, where we found <i>Pitx1</i> expression to be more highly expressed in the hindlimb compared with the forelimb during early and late morphogenesis—a result consistent to that found across other tetrapods. Expression of <i>Pitx1</i> in forelimbs has only rarely been documented, including via <i>in situ</i> hybridization in a chicken and a frog. Our findings, from both RT-qPCR and IHC, indicate that further research across a wider range of tetrapods is needed to more fully understand evolutionary variation in molecular processes underlying limb morphology

Primers designed for this study, which were used in sequencing and in QPCR for the three limb development genes and ribosomal 28S in Pogona vitticeps from Expression of a hindlimb-determining factor <i>Pitx1</i> in the forelimb of the lizard <i>Pogona vitticeps</i> during morphogenesis

With over 9000 species, squamates, which include lizards and snakes, are the largest group of reptiles and second-largest order of vertebrates, spanning a vast array of appendicular skeletal morphology. As such, they provide a promising system for examining developmental and molecular processes underlying limb morphology. Using the Central Bearded Dragon (<i>Pogona vitticeps</i>) as the primary study model, we examined limb morphometry throughout embryonic development and characterized the expression of three known developmental genes (<i>GHR, Pitx1</i> and <i>Shh</i>) from early embryonic stage through to hatchling stage via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). In this study, all genes were found to be transcribed in both the forelimb and hindlimbs of <i>P. vitticeps.</i> While the highest level of <i>GHR</i> expression occurred at the hatchling stage, <i>Pitx1</i> and <i>Shh</i> expression was greatest earlier during embryogenesis, which coincides with the onset of the differentiation between forelimb and hindlimb length. We compared our finding of <i>Pitx1</i> expression—a hindlimb-determining gene—in the forelimbs of <i>P. vitticeps</i> to that in a closely related Australian agamid lizard, <i>Ctenophorus pictus</i>, where we found <i>Pitx1</i> expression to be more highly expressed in the hindlimb compared with the forelimb during early and late morphogenesis—a result consistent to that found across other tetrapods. Expression of <i>Pitx1</i> in forelimbs has only rarely been documented, including via <i>in situ</i> hybridization in a chicken and a frog. Our findings, from both RT-qPCR and IHC, indicate that further research across a wider range of tetrapods is needed to more fully understand evolutionary variation in molecular processes underlying limb morphology

Embryonic Staging in Pogona vitticeps from Expression of a hindlimb-determining factor <i>Pitx1</i> in the forelimb of the lizard <i>Pogona vitticeps</i> during morphogenesis

With over 9000 species, squamates, which include lizards and snakes, are the largest group of reptiles and second-largest order of vertebrates, spanning a vast array of appendicular skeletal morphology. As such, they provide a promising system for examining developmental and molecular processes underlying limb morphology. Using the Central Bearded Dragon (<i>Pogona vitticeps</i>) as the primary study model, we examined limb morphometry throughout embryonic development and characterized the expression of three known developmental genes (<i>GHR, Pitx1</i> and <i>Shh</i>) from early embryonic stage through to hatchling stage via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). In this study, all genes were found to be transcribed in both the forelimb and hindlimbs of <i>P. vitticeps.</i> While the highest level of <i>GHR</i> expression occurred at the hatchling stage, <i>Pitx1</i> and <i>Shh</i> expression was greatest earlier during embryogenesis, which coincides with the onset of the differentiation between forelimb and hindlimb length. We compared our finding of <i>Pitx1</i> expression—a hindlimb-determining gene—in the forelimbs of <i>P. vitticeps</i> to that in a closely related Australian agamid lizard, <i>Ctenophorus pictus</i>, where we found <i>Pitx1</i> expression to be more highly expressed in the hindlimb compared with the forelimb during early and late morphogenesis—a result consistent to that found across other tetrapods. Expression of <i>Pitx1</i> in forelimbs has only rarely been documented, including via <i>in situ</i> hybridization in a chicken and a frog. Our findings, from both RT-qPCR and IHC, indicate that further research across a wider range of tetrapods is needed to more fully understand evolutionary variation in molecular processes underlying limb morphology

Additional uncropped IHC images for Pogona vitticeps from Expression of a hindlimb-determining factor <i>Pitx1</i> in the forelimb of the lizard <i>Pogona vitticeps</i> during morphogenesis

With over 9000 species, squamates, which include lizards and snakes, are the largest group of reptiles and second-largest order of vertebrates, spanning a vast array of appendicular skeletal morphology. As such, they provide a promising system for examining developmental and molecular processes underlying limb morphology. Using the Central Bearded Dragon (<i>Pogona vitticeps</i>) as the primary study model, we examined limb morphometry throughout embryonic development and characterized the expression of three known developmental genes (<i>GHR, Pitx1</i> and <i>Shh</i>) from early embryonic stage through to hatchling stage via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). In this study, all genes were found to be transcribed in both the forelimb and hindlimbs of <i>P. vitticeps.</i> While the highest level of <i>GHR</i> expression occurred at the hatchling stage, <i>Pitx1</i> and <i>Shh</i> expression was greatest earlier during embryogenesis, which coincides with the onset of the differentiation between forelimb and hindlimb length. We compared our finding of <i>Pitx1</i> expression—a hindlimb-determining gene—in the forelimbs of <i>P. vitticeps</i> to that in a closely related Australian agamid lizard, <i>Ctenophorus pictus</i>, where we found <i>Pitx1</i> expression to be more highly expressed in the hindlimb compared with the forelimb during early and late morphogenesis—a result consistent to that found across other tetrapods. Expression of <i>Pitx1</i> in forelimbs has only rarely been documented, including via <i>in situ</i> hybridization in a chicken and a frog. Our findings, from both RT-qPCR and IHC, indicate that further research across a wider range of tetrapods is needed to more fully understand evolutionary variation in molecular processes underlying limb morphology