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:Fire disturbances are becoming more common, more intense, and further-reaching across the globe, with consequences for ecosystem functioning. Importantly, fire can have strong effects on the soil microbiome, including community and functional changes after fire, but surprisingly little is known regarding the role of soil fire legacy in shaping responses to recent fire. To address this gap, we conducted a manipulative field experiment administering fire across 32 soils with varying fire legacies, including combinations of 1-7 historic fires and 1-33 years since most recent fire. We analyzed soil metatranscriptomes, determining for the first time how fire and fire legacy interactively affect metabolically-active soil taxa, the microbial regulation of important carbon (C), nitrogen (N) and phosphorus (P) cycling, expression of carbohydrate-cycling enzyme pathways, and functional gene co-expression networks. Experimental fire strongly downregulated fungal activity while upregulating many bacterial and archaeal phyla. Further, fire decreased soil capacity for microbial C and N cycling and P transport, and drastically rewired functional gene co-expression. Perhaps most importantly, we highlight a novel role of soil fire legacy in regulation of microbial C, N, and P responses to recent fire. We observed a greater number of functional genes responsive to the interactive effects of fire and fire legacy than those affected solely by recent fire, indicating that many functional genes respond to fire only under certain fire legacy contexts. Therefore, without incorporating fire legacy of soils, studies will miss important ways that fire shapes microbial roles in ecosystem functioning. Finally, we showed that fire caused significant downregulation of carbon metabolism and nutrient cycling genes in microbiomes under abnormal soil fire histories, producing a novel warning for the future: human manipulation of fire legacies, either indirectly through global change-induced fire intensification or directly through fire suppression, can negatively impact soil microbiome functional responses to new fires.
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: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:The analysis of transcriptomes is well-established and increasingly affordable in studies at the interface of ecology and evolution. Expression analysis of thousands of genes in parallel reveals functions and pathways involved in relevant phenotypic differentiation. The application of such methods typically involves the sacrifice of the analysed organisms, which is potentially subject to ethical and legal constraints. As an alternative to lethal sampling, transcriptome analyses can be performed using small biopsies of dispensable tissues. It has to be verified, however, to what extent such results are representative of the whole organism. Here, we use a custom microarray to compare transcriptomes of tail-clip samples with those of the remaining whole-body of fire salamander larvae (Salamandra salamandra). The microarray was calibrated using target RNA to validate the performance of each probe. We varied water temperature to test whether the thermal response in gene expression can be characterized in both types of sample. A large fraction (51 %) of the differentially expressed genes showed parallel changes for both tail clips and whole bodies in response to temperature. While sets of differentially expressed were not identical, they largely belonged to the same functional categories. The gene functions thus revealed a common thermal response of larvae irrespective of the sampled tissue. This included an overexpression of mitochondrial transcripts, an expected thermal acclimatization response of ectotherms. Hence, ecological transcriptomics based on small biopsies represent an alternative to the analysis of lethally sampled tissues in situations where the sacrifice of individuals is not an option. Larvae of European Fire salamander were exposed to two different temperatures (9°C & 17°C; n(cold) = 6, n(warm) = 6, n(total) = 12). The transcriptome response to temperature was assessed based on RNA extracted from tail tips, which can be sampled without sacrificing the individual, and based on RNA extracted from the remaining whole body. Results from the analysis of both tissues were compared.
Project description:Morphological and transcriptomic analyses reveal three discrete primary stages of postembryonic development in the common fire salamander, Salamandra salamandra