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:Joint range of motion (RoM) analyses are fundamental to our understanding of how an animal moves throughout its ecosystem. Recent technological advances allow for more detailed quantification of this RoM (e.g. including interaction of degrees of freedom) both in ex vivo joints and in vivo experiments. Both types of data have been used to draw comparisons with fossils to reconstruct locomotion. Salamanders are often used as analogues for early tetrapod locomotion; testing such hypotheses requires an in-depth analysis of salamander joint RoM. Here, we provide a detailed dataset of the ex vivo ligamentous rotational joint RoM in the hindlimb of the fire salamander Salamandra salamandra, using a new method for collecting and visualising joint RoM. We also characterise in vivo joint RoM used during walking, via scientific rotoscoping and compare the in vivo and ex vivo data. In summary, we provide (1) a new method for joint RoM data experiments and (2) a detailed analysis of both in vivo and ex vivo data of salamander hindlimbs, which can be used for comparative studies.
Project description:Phenotypic plasticity has been proposed as a mechanism facilitating the colonisation and adaptation to novel environments, such as caves. However, phenotypic plasticity in subterranean environments remains largely unexplored. Here, we test for plasticity in growth and development of fire salamander larvae (Salamandra salamandra) from subterranean and surface habitats, in response to contrasting food availability and light conditions. We hypothesized that: (i) low food availability and absence of light decrease larval growth and delay metamorphosis, (ii) light conditions mediate the effects of food availability on growth and time to metamorphosis, and (iii) larval response to contrasting light and food conditions is shaped by the habitat of origin. Our study showed that reduced food availability significantly delayed metamorphosis and slowed total length and body mass growth rates, while exposure to constant darkness slowed body mass growth rate. However, larvae slowed growth rates and increased time to metamorphosis without compromising size at metamorphosis. The effect of food availability on growth and time to metamorphosis did not change under different light conditions. Fire salamanders from subterranean and surface habitats responded differently only in relation to contrasting food availability conditions. Specifically, larvae from the surface habitat grew faster in high food conditions, while growth in larvae from the subterranean habitat was not influenced by food availability. Initial size also appeared to be an influential factor, since larger and heavier larvae grew slower, metamorphosed faster, and the size advantage was maintained in newly-metamorphosed juveniles. Overall, the results of our experiment suggest that plasticity and local adaptation favor the exploitation of aquatic subterranean habitats for breeding by fire salamanders, allowing successful development even under food shortage and day-length constraints, without compromising metamorphic size. Our findings have implications for conservation because they confirm that phenotypic plasticity plays a critical role in allowing fire salamanders to overcome altered environmental conditions.
Project description:Tonic immobility is an antipredator defence in which the prey animal remains motionless after physical contact with the predator, pretending to be dead. This behaviour has been observed among a variety of taxa but has received only little attention in amphibian larvae. During our field studies with fire salamander larvae, we observed that larvae from different habitats display tonic immobility after handling. In our study site, we find larvae in pond and stream habitats, that differ in several aspects such as their stress response and their risk-taking behaviour, likely caused by the very different habitat conditions. We measured the time that the tonic immobility behaviour was displayed but found no difference between larvae from the two habitat types. Likewise, we also found no correlation between the size of the larvae and the duration of displaying the behaviour. In conclusion, we found that fire salamander larvae show tonic immobility, but found no evidence that the different habitat conditions influence the tonic immobility behaviour.