Project description:Transcriptomes of organisms reveal differentiation associated with the use of different habitats. However, this leaves open how much of the observed differentiation can be attributed to genetic differences or to transcriptional plasticity. In this study, we disentangle causes of differential gene expression in larvae of the European fire salamander from the Kottenforst forest in Germany. Larvae inhabit permanent streams and ephemeral ponds and represent an example of a young evolutionary split associated with contrasting ecological conditions. We found ample evidence for differentiation among larvae occupying different habitats in nature with 2800 out of 11797 genes being differentially expressed based on transcriptome data from salamander sampled in their natural habitat (see GEO Series GSE100819). We then quantified transcriptional plasticity towards temperature and genetic differentiation based on controlled temperature laboratory experiments. Gene-by-environment interactions modelling revealed that 28 % of the gene expression divergence observed among samples in nature could be attributed to plasticity related to water temperature. Expression patterns of only a small number of 101 genes were affected by the genotype. Our analysis demonstrates that effects of environmental factors must be taken into account to explain variation of gene expression in salamanders in nature. Notwithstanding, it provides first evidence that genetic factors determined gene expression divergence between pond and stream ecotypes and could be involved in adaptive evolution.
Project description:A custom 8x60 k expression microarray for larvae of European fire salamander (Salamandra salamandra) was designed based on transcriptome sequencing. It is known the fact, that oligonucleotide probes differ in the binding behavior towards their target sequences. Therefore, we performed a calibration of our microarray where we assessed the binding behavior of the individual probes empirically. This information was used to normalize gene expression data measurements with the same microarray in another experiment. Please refer to the accompanying publication (Czypionka et al. 2015." Ecological transcriptomics – a non-lethal sampling approach for endangered fire salamanders" Methods in Ecology and Evolution) for more information.
Project description:A custom 8x60 k expression microarray for larvae of European fire salamander (Salamandra salamandra) was designed based on transcriptome sequencing. It is known the fact, that oligonucleotide probes differ in the binding behavior towards their target sequences. Therefore, we performed a calibration of our microarray where we assessed the binding behavior of the individual probes empirically. This information was used to normalize gene expression data measurements with the same microarray in another experiment. Please refer to the accompanying publication (Czypionka et al. 2015." Ecological transcriptomics – a non-lethal sampling approach for endangered fire salamanders" Methods in Ecology and Evolution) for more information. Labeled cRNA was prepared from Salamander larvae kept at 9°C and 17°C. A cRNA calibration pool was prepared with equimolar amounts of cRNA prepared from (a) a larvae (temperature: 9°C: source: pond KOE), (b) a larvae (temperature: 17°C: source: pond KOE), (c) a larvae (temperature: 9°C: source: stream KoGB (Klufterbach) and (d) a larvae (temperature: 17°C: source: stream KoGB (Klufterbach). See Steinfartz et al. (2007) (doi: 10.1111/j.1365-294X.2007.03490.x) for information of the source populations. Increasing amounts of labeled cRNA (75 ng, 150 ng, 300 ng, 600 ng, 1000 ng, 1400 ng, 1800 ng, 2200 ng), corresponding to (1/8, 1/4, 1/2, 1, 1 2/3, 2 1/3, 3 and 3 3/3 times the recommended amount of 600 ng) were hybridized to 8 microarrays (one microarray per dilution). The change in observed signal intensity in relation to the change in amount of labeled cRNA was used to infer the target-binding behavior of the individual probes. This information was extracted, to be used for a normalization procedure in another experiment with the same microarray (see Czypionka et al. 2015." Ecological transcriptomics – a non-lethal sampling approach for endangered fire salamanders" Methods in Ecology and Evolution). The current study provides only raw data for a calibration experiment, to validate the binding behavior of the different probes on a newly designed microarray for a non model organism (European Fire salamander). This calibration is based only on raw data. More information on targeted genes is provided in a different GEO dataset (currently submitted), where biological meaningful analysis are performed with data which are normalized based on this calibration.
Project description:Phenotypic plasticity and local adaptation via genetic change are two major mechanisms of response to dynamic environmental conditions. These mechanisms are not mutually exclusive, since genetic change can establish similar phenotypes to plasticity. This connection between both mechanisms raises the question of how much of the variation observed between species or populations is plastic and how much of it is genetic. In this study, we used a structured population of fire salamanders (Salamandra salamandra), in which two subpopulations differ in terms of physiology, genetics, mate-, and habitat preferences. Our goal was to identify candidate genes for differential habitat adaptation in this system, and to explore the degree of plasticity compared to local adaptation. We therefore performed a reciprocal transfer experiment of stream- and pond-originated salamander larvae and analyzed changes in morphology and transcriptomic profile (using species-specific microarrays). We observed that stream- and pond-originated individuals diverge in morphology and gene expression. For instance, pond-originated larvae have larger gills, likely to cope with oxygen-poor ponds. When transferred to streams, pond-originated larvae showed a high degree of plasticity, resembling the morphology and gene expression of stream-originated larvae (reversion); however the same was not found for stream-originated larvae when transferred to ponds, where the expression of genes related to reduction-oxidation processes was increased, possibly to cope with environmental stress. The lack of symmetrical responses between transplanted animals highlights the fact that the adaptations are not fully plastic and that some level of local adaptation has already occurred in this population. This study illuminates the process by which phenotypic plasticity allows local adaptation to new environments and its potential role in the pathway of incipient speciation.
Project description:Small populations are more prone to extinction if the dispersal among them is not adequately maintained by ecological connections. The degree of isolation between populations could be evaluated measuring their genetic distance, which depends on the respective geographic (isolation by distance, IBD) and/or ecological (isolation by resistance, IBR) distances. The aim of this study was to assess the ecological connectivity of fire salamander Salamandra salamandra populations by means of a landscape genetic approach. The species lives in broad-leaved forest ecosystems and is particularly affected by fragmentation due to its habitat selectivity and low dispersal capability. We analyzed 477 biological samples collected in 47 sampling locations (SLs) in the mainly continuous populations of the Prealpine and Eastern foothill lowland (PEF) and 10 SLs in the fragmented populations of the Western foothill (WF) lowland of Lombardy (northern Italy). Pairwise genetic distances (Chord distance, DC) were estimated from allele frequencies of 16 microsatellites loci. Ecological distances were calculated using one of the most promising methodology in landscape genetics studies, the circuit theory, applied to habitat suitability maps. We realized two habitat suitability models: one without barriers (EcoD) and a second one accounting for the possible barrier effect of main roads (EcoDb). Mantel tests between distance matrices highlighted how the Log-DC in PEF populations was related to log-transformed geographic distance (confirming a prevalence of IBD), while it was explained by the Log-EcoD, and particularly by the Log-EcoDb, in WF populations, even when accounting for the confounding effect of geographic distance (highlighting a prevalence of IBR). Moreover, we also demonstrated how considering the overall population, the effect of Euclidean or ecological distances on genetic distances acting at the level of a single group (PEF or WF populations) could not be detected, when population are strongly structured.
Project description:The postembryonic development of amphibians has been characterized as divided into three predominant periods, hereafter named primary developmental stages: premetamorphosis (PreM), prometamorphosis (ProM), metamorphic climax (Meta), and completion of metamorphosis (PostM), largely based on examination of anuran development. Here, we categorized the postembryonic development of larvae of a poisonous fire salamander (Salamandra salamandra) by integrating morphology and gene expression (transcriptomic) data. Morphological analysis revealed three distinct clusters suggestive of PreM, ProM, and Meta, which were confirmed in parallel by microarray-derived gene expression analysis. In total, 3,510 probes targeted transcripts differentially expressed between the clusters we identified. Genes upregulated in PreM related to organogenesis, and those upregulated in Meta underlie structural proteins and relate to development of anatomical structures and pigmentation. Biosynthesis pathways of pigments (pteridines and melanin) were upregulated during late ProM and Meta. Gas chromatographic analysis of alkaloids indicated the onset of steroidal alkaloid biosynthesis at ProM. When comparing gene expression in the fire salamander to that in other amphibians—three anurans, Xenopus laevis, X. tropicalis, and Michrohyla fissipes, and one caudate, Ambystoma mexicanum—, we identified genes with conserved expression patterns involved in basic metamorphic processes such as skin restructuring and tail fin resorption. Our results support that primary stages of postembryonic development in caudates are homologous to those of anurans, and offer a baseline for the study of the evolution of developmental modes.
Project description:Our research goal is to illustrate the potential of gene expression profiling to discriminate between polluted and non-polluted field sites and predict the presence of a specific contaminant. Using a gene expression analysis, we challenged our custom Daphnia magna cDNA microarray to determine the presence of a specific metal toxicant in blinded field samples collected from two copper mines in California. We compared the gene expression profiles from our field samples to previously established expression profiles for Cu, Cd, and Zn. The expression profiles from the Cu containing field samples clustered with the Cu specific gene expression profiles. Many of the previously discovered copper biomarkers were also differentially expressed in the field samples, suggesting that gene expression analysis is capable of producing robust biomarkers of exposure, which can be validated in field studies. In addition, our study revealed that upstream field samples containing undetectable levels of Cu caused the differential expression of only a few genes, lending support for the concept of a No Observed Transcriptional Effect Level (NOTEL). If confirmed by further studies, the NOTEL may play an important role in discriminating polluted and non-polluted sites in future monitoring efforts. Keywords: ecotoxicogenomic exposure study
Project description:Our research goal is to illustrate the potential of gene expression profiling to discriminate between polluted and non-polluted field sites and predict the presence of a specific contaminant. Using a gene expression analysis, we challenged our custom Daphnia magna cDNA microarray to determine the presence of a specific metal toxicant in blinded field samples collected from two copper mines in California. We compared the gene expression profiles from our field samples to previously established expression profiles for Cu, Cd, and Zn. The expression profiles from the Cu containing field samples clustered with the Cu specific gene expression profiles. Many of the previously discovered copper biomarkers were also differentially expressed in the field samples, suggesting that gene expression analysis is capable of producing robust biomarkers of exposure, which can be validated in field studies. In addition, our study revealed that upstream field samples containing undetectable levels of Cu caused the differential expression of only a few genes, lending support for the concept of a No Observed Transcriptional Effect Level (NOTEL). If confirmed by further studies, the NOTEL may play an important role in discriminating polluted and non-polluted sites in future monitoring efforts. Keywords: ecotoxicogenomic exposure study
Project description:Phenotypic plasticity and local adaptation via genetic change are two major mechanisms of response to dynamic environmental conditions. These mechanisms are not mutually exclusive, since genetic change can establish similar phenotypes to plasticity. This connection between both mechanisms raises the question of how much of the variation observed between species or populations is plastic and how much of it is genetic. In this study, we used a structured population of fire salamanders (Salamandra salamandra), in which two subpopulations differ in terms of physiology, genetics, mate-, and habitat preferences. Our goal was to identify candidate genes for differential habitat adaptation in this system, and to explore the degree of plasticity compared to local adaptation. We therefore performed a reciprocal transfer experiment of stream- and pond-originated salamander larvae and analyzed changes in morphology and transcriptomic profile (using species-specific microarrays). We observed that stream- and pond-originated individuals diverge in morphology and gene expression. For instance, pond-originated larvae have larger gills, likely to cope with oxygen-poor ponds. When transferred to streams, pond-originated larvae showed a high degree of plasticity, resembling the morphology and gene expression of stream-originated larvae (reversion); however the same was not found for stream-originated larvae when transferred to ponds, where the expression of genes related to reduction-oxidation processes was increased, possibly to cope with environmental stress. The lack of symmetrical responses between transplanted animals highlights the fact that the adaptations are not fully plastic and that some level of local adaptation has already occurred in this population. This study illuminates the process by which phenotypic plasticity allows local adaptation to new environments and its potential role in the pathway of incipient speciation.