Evolution at two time frames: Polymorphisms from an ancient singular divergence event fuel contemporary parallel evolution.

When environments change, populations may adapt surprisingly fast, repeatedly and even at microgeographic scales. There is increasing evidence that such cases of rapid parallel evolution are fueled by standing genetic variation, but the source of this genetic variation remains poorly understood. In the saltmarsh beetle Pogonus chalceus, short-winged 'tidal' and long-winged 'seasonal' ecotypes have diverged in response to contrasting hydrological regimes and can be repeatedly found along the Atlantic European coast. By analyzing genomic variation across the beetles' distribution, we reveal that alleles selected in the tidal ecotype are spread across the genome and evolved during a singular and, likely, geographically isolated divergence event, within the last 190 Kya. Due to subsequent admixture, the ancient and differentially selected alleles are currently polymorphic in most populations across its range, which could potentially allow for the fast evolution of one ecotype from a small number of random individuals, as low as 5 to 15, from a population of the other ecotype. Our results suggest that cases of fast parallel ecological divergence can be the result of evolution at two different time frames: divergence in the past, followed by repeated selection on the same divergently evolved alleles after admixture. These findings highlight the importance of an ancient and, likely, allopatric divergence event for driving the rate and direction of contemporary fast evolution under gene flow. This mechanism is potentially driven by periods of geographic isolation imposed by large-scale environmental changes such as glacial cycles.

Towards an evolutionary history of European-Alpine Trechus ground beetles: Species groups and wing reduction.

The evolution of flight triggered the rise of pterygote insects, but secondary flightlessness has evolved numerous times and is often associated with reduced gene flow among populations and patterns of diversification. With 85 species most of which are wing reduced, the ground beetle genus Trechus in the European Alps may be one such example. Here, we reconstructed a molecular phylogeny using 72 of these species based on mitochondrial and nuclear DNA sequences as a basis for reconstructing their evolutionary history. We rearranged the species into 20 monophyletic species groups, of which five are novel and 15 were already established but with different species allocated. Wing measurements revealed a strong tendency for wing reduction but also variation within and among species, with the few fully-winged species distributed across multiple species groups containing also wing-reduced species. Using character mapping and phylogenetic independent contrasts, we found that neither distribution area, body size, pigmentation, elevational zone, nor hygrophily explained wing status in our sample. Assessing five completely sampled clades, we inferred that each of their ancestors had most likely already been wing reduced. We discuss putative scenarios explaining this pattern and the presence of wing polymorphism across the phylogeny. One plausible scenario would be an already wing-reduced last common ancestor of all Trechus species and multiple regains of full wing length via back mutation and/or hybridisation. Alternatively and possibly more likely, the ancestors were either fully winged, with subsequent rapid and repeated wing reduction explaining the current wing-status pattern, or polymorphic, with long-term polymorphism or reselection acting on standing genetic variation explaining the recent fully-winged species. Either way, Trechus ground beetles are a promising, taxonomically and ecologically diverse system for studying the evolution of flightlessness. Areas for future research include morphological assessment of flight muscles, functional analysis of flight capability, and exploration of the mechanistic and genetic bases of wing and flight evolution.

Genomics and the origin of species.

Speciation is a fundamental evolutionary process, the knowledge of which is crucial for understanding the origins of biodiversity. Genomic approaches are an increasingly important aspect of this research field. We review current understanding of genome-wide effects of accumulating reproductive isolation and of genomic properties that influence the process of speciation. Building on this work, we identify emergent trends and gaps in our understanding, propose new approaches to more fully integrate genomics into speciation research, translate speciation theory into hypotheses that are testable using genomic tools and provide an integrative definition of the field of speciation genomics.

An Alpine ant's behavioural polymorphism: monogyny with and without internest aggression in Tetramorium alpestre.

Social structure influences animal societies on various levels (e.g., relatedness, behaviour). In ants, both the number of matings per queen and the number of queens per colony can vary strongly. While workers from both monogynous and polygynous colonies often fight fiercely, in supercolonies (an extreme form of polygyny comprising thousands of queens in spatially separated but interconnected nests), non-nestmates interact peacefully. Studies on social and behavioural polymorphism within ant species can help elucidate their influence on genetic diversity and behaviour and the factors triggering variation in social structure and behaviour. Here, we reveal a behavioural and social polymorphism comprising monogyny with and without internest aggression in Tetramorium alpestre sampled in Tyrol, Austria. The social polymorphism is based on genetic and behavioural evidence and contrasts with the supercolonial organisation known from another location in Austria (Carinthia), 150 km away. Microsatellite genotyping using eight polymorphic loci revealed monogyny-monandry and high intranest pairwise relatedness. Interestingly, various experimental one-on-one worker encounters revealed only occasional aggressive behaviour between monogynous colonies, and thus a behavioural polymorphism. Mantel tests revealed a significant negative correlation between spatial distance and relatedness, while worker behaviour was not correlated with relatedness or spatial distance. These results indicate that behaviour might be influenced by other factors - for example, the experience of workers, ecological, chemical, and/or genetic factors not characterised in this study. However, workers distinguished nestmates from non-nestmates also when aggression was lacking. We hypothesise an adaptive value of reduced aggression. We speculate that the non-aggressive and partly aggressive encounters observed represent different options in the social structure of T. alpestre, the non-aggressiveness possibly also promoting supercolony development. The social and behavioural polymorphisms observed offer opportunities to identify the factors triggering these changes and thus further explore the behavioural and social polymorphism of this ant species.

Population genetic dynamics of an invasion reconstructed from the sediment egg bank.

Biological invasions are a global issue with far-reaching consequences for single species, communities and whole ecosystems. Our understanding of modes and mechanisms of biological invasions requires knowledge of the genetic processes associated with successful invasions. In many instances, this information is particularly difficult to obtain as the initial phases of the invasion process often pass unnoticed and we rely on inferences from contemporary population genetic data. Here, we combined historic information with the genetic analysis of resting eggs to reconstruct the invasion of Daphnia pulicaria into Lower Lake Constance (LLC) in the 1970s from the resting egg bank in the sediments. We identified the invader as 'European D. pulicaria' originating from meso- and eutrophic lowland lakes and ponds in Central Europe. The founding population was characterized by extremely low genetic variation in the resting egg bank that increased considerably over time. Furthermore, strong evidence for selfing and/or biparental inbreeding was found during the initial phase of the invasion, followed by a drop of selfing rate to low levels in subsequent decades. Moreover, the increase in genetic variation was most pronounced during early stages of the invasion, suggesting additional introductions during this period. Our study highlights that genetic data covering the entire invasion process from its beginning can be crucial to accurately reconstruct the invasion history of a species. We show that propagule banks can preserve such information enabling the study of population genetic dynamics and sources of genetic variation in successful invasive populations.

Quantifying interspecific and intraspecific diversity effects on ecosystem functioning.

Rapid environmental changes result in massive biodiversity loss, with detrimental consequences for the functioning of ecosystems. Recent studies suggest that intraspecific diversity can contribute to ecosystem functioning to an extent comparable to contributions of interspecific diversity. Knowledge on the relative importance of these two sources of biodiversity is essential for predicting ecosystem consequences of biodiversity loss and will aid in the prioritization of conservation targets and implementation of management measures. However, our quantitative insights into how interspecific and intraspecific biodiversity loss affects ecosystem functioning and how the effects of these two sources of biodiversity loss on ecosystem functioning can be compared are still very limited. To facilitate such quantitative insights, we extend the interspecific Price partitioning method originally introduced by J. Fox in 2006, previously used to quantify species loss and gain effects on ecosystem functioning, to also account for the effects of intraspecific diversity loss and gain on ecosystem function. Using this extended version can yield the quantitative information required for answering research questions addressing correlations between interspecific and intraspecific diversity effects on ecosystem functioning, identifying interspecific and intraspecific groups with large effects, and assessing whether intraspecific diversity can compensate for losses in interspecific diversity. Applying this method to carefully designed experiments will provide additional insights into how biodiversity loss at different ecological levels contributes to and changes ecosystem functioning.

Anthropogenic Change and the Process of Speciation.

Anthropogenic impacts on the environment alter speciation processes by affecting both geographical contexts and selection patterns on a worldwide scale. Here we review evidence of these effects. We find that human activities often generate spatial isolation between populations and thereby promote genetic divergence but also frequently cause sudden secondary contact and hybridization between diverging lineages. Human-caused environmental changes produce new ecological niches, altering selection in diverse ways that can drive diversification; but changes also often remove niches and cause extirpations. Human impacts that alter selection regimes are widespread and strong in magnitude, ranging from local changes in biotic and abiotic conditions to direct harvesting to global climate change. Altered selection, and evolutionary responses to it, impacts early-stage divergence of lineages, but does not necessarily lead toward speciation and persistence of separate species. Altogether, humans both promote and hinder speciation, although new species would form very slowly relative to anthropogenic hybridization, which can be nearly instantaneous. Speculating about the future of speciation, we highlight two key conclusions: (1) Humans will have a large influence on extinction and "despeciation" dynamics in the short term and on early-stage lineage divergence, and thus potentially speciation in the longer term, and (2) long-term monitoring combined with easily dated anthropogenic changes will improve our understanding of the processes of speciation. We can use this knowledge to preserve and restore ecosystems in ways that promote (re-)diversification, increasing future opportunities of speciation and enhancing biodiversity.

Reversed evolution of grazer resistance to cyanobacteria.

Exploring the capability of organisms to cope with human-caused environmental change is crucial for assessing the risk of extinction and biodiversity loss. We study the consequences of changing nutrient pollution for the freshwater keystone grazer, Daphnia, in a large lake with a well-documented history of eutrophication and oligotrophication. Experiments using decades-old genotypes resurrected from the sediment egg bank revealed that nutrient enrichment in the middle of the 20th century, resulting in the proliferation of harmful cyanobacteria, led to the rapid evolution of grazer resistance to cyanobacteria. We show here that the subsequent reduction in nutrient input, accompanied by a decrease in cyanobacteria, resulted in the re-emergence of highly susceptible Daphnia genotypes. Expression and subsequent loss of grazer resistance occurred at high evolutionary rates, suggesting opposing selection and that maintaining resistance was costly. We provide a rare example of reversed evolution of a fitness-relevant trait in response to relaxed selection.

Hybridization Dynamics and Extensive Introgression in the Daphnia longispina Species Complex: New Insights from a High-Quality Daphnia galeata Reference Genome.

Hybridization and introgression are recognized as an important source of variation that influence adaptive processes; both phenomena are frequent in the genus Daphnia, a keystone zooplankton taxon in freshwater ecosystems that comprises several species complexes. To investigate genome-wide consequences of introgression between species, we provide here the first high-quality genome assembly for a member of the Daphnia longispina species complex, Daphnia galeata. We further resequenced 49 whole genomes of three species of the complex and their interspecific hybrids both from genotypes sampled in the water column and from single resting eggs extracted from sediment cores. Populations from habitats with diverse ecological conditions offered an opportunity to study the dynamics of hybridization linked to ecological changes and revealed a high prevalence of hybrids. Using phylogenetic and population genomic approaches, we provide first insights into the intra- and interspecific genome-wide variability in this species complex and identify regions of high divergence. Finally, we assess the length of ancestry tracts in hybrids to characterize introgression patterns across the genome. Our analyses uncover a complex history of hybridization and introgression reflecting multiple generations of hybridization and backcrossing in the Daphnia longispina species complex. Overall, this study and the new resources presented here pave the way for a better understanding of ancient and contemporary gene flow in the species complex and facilitate future studies on resting egg banks accumulating in lake sediment.

Optimized and affordable high-throughput sequencing workflow for preserved and nonpreserved small zooplankton specimens.

Genomic analysis of hundreds of individuals is increasingly becoming standard in evolutionary and ecological research. Individual-based sequencing generates large amounts of valuable data from experimental and field studies, while using preserved samples is an invaluable resource for studying biodiversity in remote areas or across time. Yet, small-bodied individuals or specimens from collections are often of limited use for genomic analyses due to a lack of suitable extraction and library preparation protocols for preserved or small amounts of tissues. Currently, high-throughput sequencing in zooplankton is mostly restricted to clonal species, that can be maintained in live cultures to obtain sufficient amounts of tissue, or relies on a whole-genome amplification step that comes with several biases and high costs. Here, we present a workflow for high-throughput sequencing of single small individuals omitting the need for prior whole-genome amplification or live cultures. We establish and demonstrate this method using 27 species of the genus Daphnia, aquatic keystone organisms, and validate it with small-bodied ostracods. Our workflow is applicable to both live and preserved samples at low costs per sample. We first show that a silica-column based DNA extraction method resulted in the highest DNA yields for nonpreserved samples while a precipitation-based technique gave the highest yield for ethanol-preserved samples and provided the longest DNA fragments. We then successfully performed short-read whole genome sequencing from single Daphnia specimens and ostracods. Moreover, we assembled a draft reference genome from a single Daphnia individual (>50× coverage) highlighting the value of the workflow for non-model organisms.

Large geographic distance versus small DNA barcode divergence: Insights from a comparison of European to South Siberian Lepidoptera.

Spanning nearly 13,000 km, the Palearctic region provides an opportunity to examine the level of geographic coverage required for a DNA barcode reference library to be effective in identifying species with broad ranges. This study examines barcode divergences between populations of 102 species of Lepidoptera from Europe and South Siberia, sites roughly 6,000 km apart. While three-quarters of these species showed divergence between their Asian and European populations, these divergence values ranged between 0-1%, distinctly less than the distance to the Nearest-Neighbor species in all but a few cases. Our results suggest that further taxonomic studies may be required for 16 species that showed either extremely low interspecific or high intraspecific variation. For example, seven species pairs showed low or no barcode divergence, but four of these cases are likely to reflect taxonomic over-splitting while the others involve species pairs that are either young or show evidence for introgression. Conversely, some of the nine species with deep intraspecific divergence at varied spatial levels may include overlooked species. Although these 16 cases require further investigation, our overall results indicate that barcode reference libraries based on records from one locality can be very effective in identifying specimens across an extensive geographic area.

Natural Selection and Genetic Diversity in the Butterfly Heliconius melpomene.

A combination of selective and neutral evolutionary forces shape patterns of genetic diversity in nature. Among the insects, most previous analyses of the roles of drift and selection in shaping variation across the genome have focused on the genus Drosophila A more complete understanding of these forces will come from analyzing other taxa that differ in population demography and other aspects of biology. We have analyzed diversity and signatures of selection in the neotropical Heliconius butterflies using resequenced genomes from 58 wild-caught individuals of Heliconius melpomene and another 21 resequenced genomes representing 11 related species. By comparing intraspecific diversity and interspecific divergence, we estimate that 31% of amino acid substitutions between Heliconius species are adaptive. Diversity at putatively neutral sites is negatively correlated with the local density of coding sites as well as nonsynonymous substitutions and positively correlated with recombination rate, indicating widespread linked selection. This process also manifests in significantly reduced diversity on longer chromosomes, consistent with lower recombination rates. Although hitchhiking around beneficial nonsynonymous mutations has significantly shaped genetic variation in H. melpomene, evidence for strong selective sweeps is limited overall. We did however identify two regions where distinct haplotypes have swept in different populations, leading to increased population differentiation. On the whole, our study suggests that positive selection is less pervasive in these butterflies as compared to fruit flies, a fact that curiously results in very similar levels of neutral diversity in these very different insects.

A mixture of environmental organic contaminants in lake sediments affects hatching from Daphnia resting eggs.

Despite the relevance of resting eggs for ecology and evolution of many aquatic organisms and their exposure to contaminants accumulating in sediments, ecotoxicological studies using resting eggs are vastly underrepresented. The authors established a method to perform exposure assays with resting eggs produced by the Daphnia longispina species complex, key species in large lake ecosystems. A mixture of organic contaminants previously detected in sediments of Lake Greifensee was selected to test the potential effect of organic contaminants present in sediments on the hatching process. Resting eggs were exposed to a mix of 10 chemicals, which included corrosion inhibitors, biocides, pesticides, and personal care products, for a period of 15 d. Using an automated counting software, the authors found a significant increase in hatching success in the exposed resting eggs compared with controls. Such an effect has not yet been reported from ecotoxicological assays with resting eggs. Possible mechanistic explanations as well as the potential implications on the ecology and evolution of aquatic species that rely on a resting egg banks are discussed. Observed increased mortality and developmental abnormalities for hatchlings in the exposure treatments can be explained by toxic contaminant concentrations. The results of the present study highlight the need for additional studies assessing the effects of organic contaminants on resting egg banks and aquatic ecosystems.

Linking oxygen to time: the bidirectional interaction between the hypoxic signaling pathway and the circadian clock.

The circadian clock and the hypoxic signaling pathway play critical roles in physiological homeostasis as well as in tumorgenesis. Interactions between both pathways have repeatedly been reported for mammals during the last decade, the molecular basis, though, has not been identified so far. Expression levels of oxygen-regulated and circadian clock genes in zebrafish larvae (Danio rerio) and zebrafish cell lines were significantly altered under hypoxic conditions. Thus, long-term hypoxic incubation of larvae resulted in a dampening of the diurnal oscillation amplitude of the period1 gene expression starting only several hours after start of the hypoxic incubation. A significant decrease in the amplitude of the period1 circadian oscillation in response to hypoxia and in response to the hypoxic mimic CoCl2 was also observed using a zebrafish luciferase reporter cell line in constant darkness. In addition, activity measurements of zebrafish larvae using an infrared-sensitive camera demonstrated the loss of their usual circadian activity pattern under hypoxic conditions. To explore the functional basis of the observed cross-talk between both signaling pathways ChIP assays were performed. Increasing with the duration of hypoxia, a nearly 4-fold occupancy of hypoxia-inducible factor 1 (Hif-1α) at two specific E-box binding sites located in the period1 gene control region was shown, demonstrating therewith the transcriptional co-regulation of the core clock gene by the major transcription factor of the hypoxic pathway. On the other hand, circadian transgenic zebrafish cells, simulating a repressed or an overstimulated circadian clock, modified gene transcription levels of oxygen-regulated genes such as erythropoietin and vascular endothelial growth factor 165 and altered the hypoxia-induced increase in Hif-1α protein concentration. In addition, the amount of Hif-1α protein accumulated during the hypoxic response was shown to depend on the time of the day, with one maximum during the light phase and a second one during the dark phase. The direct binding of Hif-1α to the period1 gene control region provides a mechanistic explanation for the repeatedly observed interaction between hypoxia and the circadian clock. The cross-talk between both major signaling pathways was shown for the first time to be bidirectional and may provide the advantage of orchestrating a broad range of genes and metabolic pathways to cope with altered oxygen availabilities.