Local adaptation in the presence of gene flow in Patagonian and Antarctic Nacella limpets: a multimarker genetic and physiological approach

The understanding of how speciation works in the ocean has always challenged marine biologists because the marine realm is characterized by the absence of strict barriers to gene flow over large geographical scales. The impact of natural selection as a source of speciation in the marine system has also been considered to be low because of the very high gene flow overriding the effects of locally varying selective pressures. In a top-down approach I analyzed large-scale impacts of emerging geographical barriers and small-scale influences of biogeographic patterning and local adaptations on the evolutionary history of Antarctic and South American Nacella limpets. A large set of genetic and physiological, neutral and selected markers was applied. The results show that natural selection and local adaptation are much more important drivers of marine speciation than previously believed. The application of genetic markers under selection revealed how natural selection is capable to maintain adaptive divergence in parts of a genome to ensure adaptability to environmental constraints even in high gene flow environments

STAMP: Extensions to the STADEN sequence analysis package for high throughput interactive microsatellite marker design

BackgroundMicrosatellites (MSs) are DNA markers with high analytical power, which are widely used in population genetics, genetic mapping, and forensic studies. Currently available software solutions for high-throughput MS design (i) have shortcomings in detecting and distinguishing imperfect and perfect MSs, (ii) lack often necessary interactive design steps, and (iii) do not allow for the development of primers for multiplex amplifications. We present a set of new tools implemented as extensions to the STADEN package, which provides the backbone functionality for flexible sequence analysis workflows. The possibility to assemble overlapping reads into unique contigs (provided by the base functionality of the STADEN package) is important to avoid developing redundant markers, a feature missing from most other similar tools.ResultsOur extensions to the STADEN package provide the following functionality to facilitate microsatellite (and also minisatellite) marker design: The new modules (i) integrate the state-of-the-art tandem repeat detection and analysis software PHOBOS into workflows, (ii) provide two separate repeat detection steps with different search criteria one for masking repetitive regions during assembly of sequencing reads and the other for designing repeat-flanking primers for MS candidate loci, (iii) incorporate the widely used primer design program PRIMER3 into STADEN workflows, enabling the interactive design and visualization of flanking primers for microsatellites, and (iv) provide the functionality to find optimal locus- and primer pair combinations for multiplex primer design. Furthermore, our extensions include a module for storing analysis results in an SQLite database, providing a transparent solution for data access from within as well as from outside of the STADEN Package.ConclusionThe STADEN package is enhanced by our modules into a highly flexible, high-throughput, interactive tool for conventional and multiplex microsatellite marker design. It gives the user detailed control over the workflow, enabling flexible combinations of manual and automated analysis steps. The software is available under the OpenBSD License [1,2]. The high efficiency of our automated marker design workflow has been confirmed in three microsatellite development projects

Local adaptation in the presence of gene flow in Patagonian and Antarctic Nacella limpets

The classical belief that speciation needs geographical isolation has raised the question how the huge amount of biodiversity can be explained in an open environment like the ocean that is characterized by the absence of strict barriers to gene flow over large geographical scales. Many theoretical approaches have contested that classical belief showing how speciation can take place in the background of gene flow rendering natural selection as a potent evolutionary force. However, most of the empirical population genetic studies aiming at speciation processes apply neutral molecular markers which do not respond to selective forces and therefore do not allow for statements on the actual roles of selection causing ecologically based barriers to gene flow. The present study‘s superordinate aim was to reconstruct the evolutionary history of South American and Antarctic patellogastropods of the genus Nacella. The central aspects of the thesis were the investigation of the actual roles of gene flow barriers and natural selection in processes causing speciation and population divergence. In order to fulfill this task a wide range of markers, both neutrally evolving and under selection, was applied to reveal gene flow patterns and local adaptation. The ACC is one of the most prominent physical barriers in the ocean. Its origin around 35 Ma before present should therefore be reflected in the timing of the divergence of Nacella. Molecular dating approaches carried out in this thesis with three different nuclear and mitochondrial genes (COI, 16S, 18S) revealed the timing of speciation of Nacella to be much younger than the proposed onset of the ACC (~ 10 Ma). Gene flow must, therefore, have been possible after the ACC had already been fully established until the Miocene 10 Ma ago. This time coincides with the second major cooling of Antarctica that led to the glaciation of the Antarctic Peninsula and most likely increased the selection for physiological and genetic adaptation on both sides of the Drake Passage. The present example highlights that the mere occurrence of the ACC alone was not sufficient to promote speciation between Antarctic and South American species. It most likely needed the additional influence of natural selection summoned by climatic changes. 135 Summary . A more detailed view on the impact of natural selection was obtained by population genetic studies of the two South American species N. magellanica and N. deaurata. They largely occur in sympatry but exhibit differences in vertical zonation along the tidal gradient. N. magellanica mainly inhabits the shallow intertidal areas and is exposed to tidal emersion twice a day. N. deaurata occurs in the deeper subtidal that is normally not heavily affected by the tidal cycles. Neutral markers (COI and seven microsatellites) reveal Southern South America to be a high gene flow environment without any genetic divergence between both morphotypes rendering them as morphotypes caused by phenotypic plasticity. The addition of genetic markers under selection (Hsp70) revealed a deep divergence between the two morphotypes. These contradicting results show that divergent selection in sympatry can cause ecologically important genomic regions to resist gene exchange, whereas in other parts of the genome gene flow continues. The genetic divergence in Hsp70 has most likely established in phases of geographical isolation of populations on Atlantic and Pacific sides of South America summoned by glacial maxima. Upon secondary contact after the Last Glacial Maximum had ended, gene flow between those populations was not restricted as seen in neutral markers. The divergence in Hsp70, however, displays how divergent selection maintains adaptive divergence in parts of the genomes in order to ensure adaptability to the environmental constraints along a tidal gradient. Physiological experiments in which individuals of N. magellanica and N. deaurata were exposed to air furthermore revealed some of the potent drivers of ecological selection that maintain genetically disrupted gene pools. The differences in vertical zonation are reflected in lower adaptability to desiccation stress and elevated temperatures summoned by tidal emersion as seen in higher heat-shock response (HSR) and enzymatic antioxidant defense levels in subtidal N. deaurata. The present study highlights the complex interplay of geographical isolation and natural selection in the evolutionary history of Nacella. The addition of genetic markers under selection provides a powerful tool to understand the importance of natural selection and local adaption as common evolutionary forces that are largely undetected by neutral marker approaches

Ecological comparison of cellular stress responses among populations – normalizing RT-qPCR values to investigate differential environmental adaptations

Background: Rising temperatures and other environmental factors influenced by global climate change can cause increased physiological stress for many species and lead to range shifts or regional population extinctions. To advance the understanding of species’ response to change and establish links between individual and ecosystem adaptations, physiological reactions have to be compared between populations living in different environments. Although changes in expression of stress genes are relatively easy to quantify, methods for reliable comparison of the data remain a contentious issue. Using normalization algorithms and further methodological considerations, we compare cellular stress response gene expression levels measured by RT-qPCR after air exposure experiments among different subpopulations of three species of the intertidal limpet Nacella. Results: Reference gene assessment algorithms reveal that stable reference genes can differ among investigated populations and / or treatment groups. Normalized expression values point to differential defense strategies to air exposure in the investigated populations, which either employ a pronounced cellular stress response in the inducible Hsp70 forms, or exhibit a comparatively high constitutive expression of Hsps (heat shock proteins) while showing only little response in terms of Hsp induction. Conclusions: This study serves as a case study to explore the methodological prerequisites of physiological stress response comparisons among ecologically and phylogenetically different organisms. To improve the reliability of gene expression data and compare the stress responses of subpopulations under potential genetic divergence, reference gene stability algorithms are valuable and necessary tools. As the Hsp70 isoforms have been shown to play different roles in the acute stress responses and increased constitutive defenses of populations in their different habitats, these comparative studies can yield insight into physiological strategies of adaptation to environmental stress and provide hints for the prudent use of the cellular stress response as a biomarker to study environmental stress and stress adaptation of populations under changing environmental conditions

Lokale Anpassung in der Gegenwart von Genfluss in patagonischen und antarktischen Napfschnecken der Gattung Nacella: ein genetischer und physiologischer Ansatz

The understanding of how speciation works in the ocean has always challenged marine biologists because the marine realm is characterized by the absence of strict barriers to gene flow over large geographical scales. The impact of natural selection as a source of speciation in the marine system has also been considered to be low because of the very high gene flow overriding the effects of locally varying selective pressures. In a top-down approach I analyzed large-scale impacts of emerging geographical barriers and small-scale influences of biogeographic patterning and local adaptations on the evolutionary history of Antarctic and South American Nacella limpets. A large set of genetic and physiological, neutral and selected markers was applied. The results show that natural selection and local adaptation are much more important drivers of marine speciation than previously believed. The application of genetic markers under selection revealed how natural selection is capable to maintain adaptive divergence in parts of a genome to ensure adaptability to environmental constraints even in high gene flow environments

Formic Acid-Based Fischer–Tropsch Synthesis for Green Fuel Production from Wet Waste Biomass and Renewable Excess Energy

While the production of hydrocarbons by Fischer–Tropsch synthesis (FTS) is a widely recognized, yet technically quite complex way to transform biomass via syngas (mostly from biomass gasification) into liquid fuels, we here present an alternative route transforming biomass first into formic acid (FA) followed by syngas formation by decomposition of FA and finally FTS using regenerative hydrogen (or if needed H$_{2}$ from the stored FA) to balance the C:H ratio. The new method builds on the recently developed, selective oxidation of biomass to formic acid using Keggin-type polyoxometalates of the general formula (H$_{3+x}$[PV$_{x}$Mo$_{12–x}$O$_{40}$]) as homogeneous catalysts, oxygen as the oxidant, and water as the solvent. This method is able to transform a wide range of complex and wet biomass mixtures into FA as the sole liquid product at mild reaction conditions (90 °C, 20–30 bar O$_{2}$). We propose to convert FA with hydrogen from water electrolysis—the electrolysis step producing also the oxygen for the biomass oxidation to FA—to green hydrocarbon fuels using a typical Co-based FT catalyst

Isolation and characterization of eight polymorphic microsatellite markers from South American limpets of the Nacella species complex

In this study we provide eight polymorphic microsatellite markers for the two South American patel- logastropods Nacella magellanica and N. deaurata. Allelic diversity ranged from 5 to 57 alleles per locus. Observed heterozygosity varied between 0.1 and 0.98. Three of the four loci designed for N. magellanica cross-amplified also with N. deaurata, and two loci vice versa. Six of the microsatellites successfully cross-amplified with the sister taxon N. mytilina. This set of microsatellites provides a suitable tool for population genetic and phylogeographic studies