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Diverse phenotypic and genetic responses to short-term selection in evolving Escherichia coli populations

Beneficial mutations fuel adaptation by altering phenotypes that enhance the fit of organisms to their environment. However, the phenotypic effects of mutations often depend on ecological context, making the distribution of effects across multiple environments essential to understanding the true nature of beneficial mutations. Studies that address both the genetic basis and ecological consequences of adaptive mutations remain rare. Here, we characterize the direct and pleiotropic fitness effects of a collection of 21 first-step beneficial mutants derived from naive and adapted genotypes used in a long-term experimental evolution of Escherichia coli. Whole-genome sequencing was used to identify most beneficial mutations. In contrast to previous studies, we find diverse fitness effects of mutations selected in a simple environment and few cases of genetic parallelism. The pleiotropic effects of these mutations were predominantly positive but some mutants were highly antagonistic in alternative environments. Further, the fitness effects of mutations derived from the adapted genotypes were dramatically reduced in nearly all environments. These findings suggest that many beneficial variants are accessible from a single point on the fitness landscape, and the fixation of alternative beneficial mutations may have dramatic consequences for niche breadth reduction via metabolic erosion.

Evolutionary Biology

Speciation driven by hybridization and chromosomal plasticity in a wild yeast

Hybridization is recognized as a powerful mechanism of speciation and a driving force in generating biodiversity. However, only few multicellular species, limited to a handful of plants and animals, have been shown to fulfill all the criteria of homoploid hybrid speciation. This lack of evidence could lead to the misconception that speciation by hybridization has a limited role in eukaryotes, particularly in single-celled organisms. Laboratory experiments have revealed that fungi such as budding yeasts can rapidly develop reproductive isolation and novel phenotypes through hybridization, showing that in principle homoploid speciation could occur in nature. Here we report a case of homoploid hybrid speciation in natural populations of the budding yeast Saccharomyces paradoxus inhabiting the North American forests. We show that the rapid evolution of chromosome architecture and an ecological context that led to secondary contact between nascent species drove the formation of an incipient hybrid species with a potentially unique ecological niche.\n\nOne Sentence SummaryChromosomal rearrangements and hybridization between two yeast lineages drive hybrid speciation after secondary contact.

Genomics

Para-allopatry in hybridizing fire-bellied toads (Bombina bombina and B. variegata): inference from transcriptome-wide coalescence analyses

Ancient origins, profound ecological divergence and extensive hybridization make the fire-bellied toads Bombina bombina and B. variegata (Anura: Bombinatoridae) an intriguing test case of ecological speciation. Narrow Bombina hybrid zones erect barriers to neutral introgression whose strength has been estimated previously. We test this prediction by inferring the rate of gene exchange between pure populations on either side of the intensively studied Krakow transect. We developed a software pipeline to extract high confidence sets of orthologous genes from de novo transcriptome assemblies, fitted a range of divergence models to these data and assessed their relative support with analytic likelihoods calculations. There was clear evidence for post-divergence gene flow, but, as expected, no perceptible signal of recent introgression via the nearby hybrid zone. The analysis of two additional Bombina taxa (B. v. scabra and B. orientalis) validated our parameter estimates against a larger set of prior expectations. Despite substantial cumulative introgression over millions of years, adaptive divergence of the hybridizing taxa is essentially unaffected by their lack of reproductive isolation. Extended distribution ranges also buffer them against small-scale environmental perturbations that have been shown to reverse the speciation process in other, more recent ecotypes.

Evolutionary Biology

Changes in the relative abundance of two Saccharomyces species from oak forests to wine fermentations

Saccharomyces cerevisiae and its sibling species S. paradoxus are known to inhabit temperate arboreal habitats across the globe. Despite their sympatric distribution in the wild, S. cerevisiae is predominantly associated with human fermentations. The apparent ecological differentiation of these species is particularly striking in Europe where S. paradoxus is abundant in forests and S. cerevisiae is abundant in vineyards. However, ecological differences may be confounded with geographic differences in species abundance. To compare the distribution and abundance of these two species we isolated Saccharomyces strains from over 1,200 samples taken from vineyard and forest habitats in Slovenia. We isolated numerous strains of S. cerevisiae and S. paradoxus as well as small number of S. kudriavzevii strains from both vineyard and forest environments. We find S. cerevisiae less abundant than S. paradoxus on oak trees within and outside the vineyard, but more abundant on grapevines and associated substrates. Analysis of the uncultured microbiome shows that both S. cerevisiae and S. paradoxus are rare species in soil and bark samples, but can be much more common in grape must. In contrast to S. paradoxus, European strains of S. cerevisiae have acquired multiple traits thought to be important for life in the vineyard and dominance of wine fermentations. We conclude that S. cerevisiae and S. paradoxus currently share both vineyard and non-vineyard habitats in Slovenia and we discuss factors relevant to their global distribution and relative abundance.

Microbiology

Evaluation of TagSeq, a reliable low-cost alternative for RNAseq

RNAseq is a relatively new tool for ecological genetics that offers researchers insight into changes in gene expression in response to a myriad of natural or experimental conditions. However, standard RNAseq methods (e.g., Illumina TruSeq(R) or NEBNext(R)) can be cost prohibitive, especially when study designs require large sample sizes. Consequently, RNAseq is often underused as a method, or is applied to small sample sizes that confer poor statistical power. Low cost RNAseq methods could therefore enable far greater and more powerful applications of transcriptomics in ecological genetics and beyond. Standard mRNAseq is costly partly because one sequences portions of the full length of all transcripts. Such whole-mRNA data is redundant for estimates of relative gene expression. TagSeq is an alternative method that focuses sequencing effort on mRNAs 3-prime end, thereby reducing the necessary sequencing depth per sample, and thus cost. Here we present a revised TagSeq protocol, and compare its performance against NEBNext(R), the gold-standard whole mRNAseq method. We built both TagSeq and NEBNext(R) libraries from the same biological samples, each spiked with control RNAs. We found that TagSeq measured the control RNA distribution more accurately than NEBNext(R), for a fraction of the cost per sample ({bsim}10%). The higher accuracy of TagSeq was particularly apparent for transcripts of moderate to low abundance. Technical replicates of TagSeq libraries are highly correlated, and were correlated with NEBNext(R) results. Overall, we show that our modified TagSeq protocol is an efficient alternative to traditional whole mRNAseq, offering researchers comparable data at greatly reduced cost.

Genomics

Geographic cline analysis as a tool for studying genome-wide variation: a case study of pollinator-mediated divergence in a monkeyflower

A major goal of speciation research is to reveal the genomic signatures that accompany the speciation process. Genome scans are routinely used to explore genome-wide variation and identify highly differentiated loci that may contribute to ecological divergence, but they do not incorporate spatial, phenotypic, or environmental data that might enhance outlier detection. Geographic cline analysis provides a potential framework for integrating diverse forms of data in a spatially-explicit framework, but it has not been used to study genome-wide patterns of divergence. Aided by a first-draft genome assembly, we combine an FCT scan and geographic cline analysis to characterize patterns of genome-wide divergence between divergent pollination ecotypes of Mimulus aurantiacus. FCT analysis of 58,872 SNPs generated via RADseq revealed little ecotypic differentiation (mean FCT = 0.041), though a small number of loci were moderately to highly diverged. Consistent with our previous results from the gene MaMyb2, which contributes to differences in flower color, 130 loci have cline shapes that recapitulate the spatial pattern of trait divergence, suggesting that they reside in or near the genomic regions that contribute to pollinator isolation. In the narrow hybrid zone between the ecotypes, extensive admixture among individuals and low linkage disequlibrium between markers indicate that outlier loci are scattered throughout the genome, rather than being restricted to one or a few regions. In addition to revealing the genomic consequences of ecological divergence in this system, we discuss how geographic cline analysis is a powerful but under-utilized framework for studying genome-wide patterns of divergence.

Genomics

Common methods for fecal sample storage in field studies yield consistent signatures of individual identity in microbiome sequencing data

Field studies of wild vertebrates are frequently associated with extensive collections of banked fecal samples, which are often collected from known individuals and sometimes also sampled longitudinally across time. Such collections represent unique resources for understanding ecological, behavioral, and phylogenetic effects on the gut microbiome, especially for species of particular conservation concern. However, we do not understand whether sample storage methods confound the ability to investigate interindividual variation in gut microbiome profiles. This uncertainty arises in part because comparisons across storage methods to date generally include only a few ([≤]5) individuals, or analyze pooled samples. Here, we used n=52 samples from 13 rhesus macaque individuals to compare immediate freezing, the gold standard of preservation, to three methods commonly used in vertebrate field studies: storage in ethanol, lyophilization following ethanol storage, and storage in RNAlater. We found that the signature of individual identity consistently outweighed storage effects: alpha diversity and beta diversity measures were significantly correlated across methods, and while samples often clustered by donor, they never clustered by storage method. Provided that all analyzed samples are stored the same way, banked fecal samples therefore appear highly suitable for investigating variation in gut microbiota. Our results open the door to a much-expanded perspective on variation in the gut microbiome across species and ecological contexts.

Genomics

Triclosan Exposure is Associated with Rapid Restructuring of the Microbiome in Adult Zebrafish

Growing evidence indicates that disrupting the microbial community that comprises the intestinal tract, known as the gut microbiome, can contribute to the development or severity of disease. As a result, it is important to discern the agents responsible for microbiome disruption. While animals are frequently exposed to a diverse array of environmental chemicals, little is known about their effects on gut microbiome stability and structure. Here, we demonstrate how zebrafish can be used to glean insight into the effects of environmental chemical exposure on the structure and ecological dynamics of the gut microbiome. Specifically, we exposed forty-five adult zebrafish to triclosan-laden food for four or seven days or a control diet, and analyzed their microbial communities using 16S rRNA amplicon sequencing. Triclosan exposure was associated with rapid shifts in microbiome structure and diversity. We find evidence that several operational taxonomic units (OTUs) associated with the family Enterobacteriaceae appear to be susceptible to triclosan exposure, while OTUs associated with the genus Pseudomonas appeared to be more resilient and resistant to exposure. We also found that triclosan exposure is associated with topological alterations to microbial interaction networks and results in an overall increase in the number of negative interactions per microbe in these networks. Together these data indicate that triclosan exposure results in altered composition and ecological dynamics of microbial communities in the gut. Our work demonstrates that because zebrafish afford rapid and inexpensive interrogation of a large number of individuals, it is a useful experimental system for the discovery of the gut microbiomes interaction with environmental chemicals.

Microbiology

Nomadic Lifestyle of Lactobacillus plantarum Revealed by Comparative Genomics of 54 strains Isolated from Different Niches

The ability of many bacteria to adapt to diverse environmental conditions is well known. Recent research has linked the process of bacterial adaptation to a niche to changes in the genome content and size, showing that many bacterial genomes reflect the constraints imposed by their habitat. However, some highly versatile bacteria are found in diverse niches that almost share nothing in common. Lactobacillus plantarum is a lactic acid bacterium that is found in a large variety of niches. With the aim of unravelling the link between genome evolution and ecological versatility of L. plantarum, we analysed the genomes of 54 L. plantarum strains isolated from different environments. Phylogenomic analyses coupled with the study of genetic functional divergence and gene-trait matching analysis revealed a mixed distribution of the strains, which was uncoupled from their environmental origin. Our findings demonstrate the high complexity of L. plantarum evolution, revealing the absence of specific genomic signatures marking adaptations of this species towards the diverse habitats it is associated with. This suggests fundamentally similar and parallel trends of genome evolution in L. plantarum, which occur in a manner that is apparently uncoupled from ecological constraint and reflects the nomadic lifestyle of this species.

Genomics

Characterization of a Male Reproductive Transcriptome for Peromyscus eremicus (Cactus mouse)

AbstractRodents of the genus Peromyscus have become increasingly utilized models for investigations into adaptive biology. This genus is particularly powerful for research linking genetics with adaptive physiology and behaviors, and recent research has capitalized on the unique opportunities afforded by the ecological diversity of these rodents. However, well characterized genomic and transcriptomic data is intrinsic to explorations of the genetic architecture responsible for ecological adaptations. This study characterizes a reproductive transcriptome of male Peromyscus eremicus (Cactus mouse), a desert specialist with extreme physiological adaptations to water limitation. We describe a reproductive transcriptome comprising three tissues in order to expand upon existing research in this species and to facilitate further studies elucidating the genetic basis of potential desert adaptations in male reproductive physiology.

Genomics

High-Throughput Sequencing of Transposable Elements Insertions Provides Evidence for Adaptive Evolution of the Invasive Asian Tiger Mosquito Towards Temperate Environments

Invasive species represent unique opportunities to evaluate the role of local adaptation during colonization of new environments. Among these species, the Asian tiger mosquito, Aedes albopictus, is a threatening vector of several human viral diseases, including dengue and chikungunya, and raises concerns about the Zika fever. Its broad presence in both temperate and tropical environments has been considered the reflection of great \"ecological plasticity\". However, no study has been conducted to assess the role of adaptive evolution in the ecological success of Ae. albopictus at the molecular level. In the present study, we performed a genomic scan to search for potential signatures of selection leading to local adaptation in one-hundred-forty field-collected mosquitoes from native populations of Vietnam and temperate invasive populations of Europe. High-throughput genotyping of transposable element insertions led to the discovery of more than 120 000 polymorphic loci, which, in their great majority, revealed a virtual absence of structure between the bio-geographic areas. Nevertheless, 92 outlier loci showed a high level of differentiation between temperate and tropical populations. The majority of these loci segregates at high insertion frequencies among European populations, indicating that this pattern could have been caused by recent adaptive evolution events in temperate areas. An analysis of the overlapping and neighboring genes highlighted several candidates, including diapause, lipid and juvenile hormone pathways.

Genomics

Different speciation types meet in a Mediterranean genus: the biogeographic history of Cymbalaria (Plantaginaceae)

Cymbalaria is a Mediterranean genus including ten species and six subspecies growing on rocky habitats. A very fragmented distribution, the different ecologic preferences of partially sympatric species and the presence of different ploidy levels, suggest the role of allopatric, sympatric ecological and polyploid speciation in its evolution. The aims of this study are to verify the monophyly and to reconstruct the phylogenetic relationships of Cymbalaria, to infer its biogeographic history by estimating the lineage divergence dates and the ancestral areas of distribution, and to discuss the role of different types of speciation. To address these issues, we constructed a complete phylogeny of the genus with ITS, 3ETS, ndhF and rpl32-trnL sequences. A time-calibrated phylogeny and an ancestral-area estimation were obtained from the nrDNA data. The evidence supported the genus Cymbalaria as monophyletic. It originated ca. 5 Ma and three lineages segregated rapidly, one with the single extant taxa Cymbalaria microcalyx subsp. microcalyx and the other two corresponding to western and central-eastern species, respectively. The main diversification events occurred after the onset of the Mediterranean climate and during Pleistocene oscillations. Founder-event and sympatric speciation were supported by the biogeographic analyses, and chromosome data combined with our phylogeny supported at least two polyploidization events. We observed that the consequences of physical barriers were different amongst the different species.\n\nSupplementary MaterialElectronic Supplement (Figure S1, Table S1) are available in the Supplementary Data section of the online version of this article (http://www.ingentaconnect.com/content/iapt/tax).

Plant Biology

Diversity of Sericostomatidae (Trichoptera) caddisflies in the Montseny mountain range

Biodiversity is under threat by the ongoing global change and especially freshwater ecosystems are under threat by intensified land use, water abstraction and other anthropogenic stressors. In order to monitor the impacts that stressors have on freshwater biodiversity, it is important to know the current state of ecosystems and species living in them. This is often hampered by lacking knowledge on species and genetic diversity due to the fact that many taxa are complexes of several morphologically cryptic species. Lacking knowledge on species identity and ecology can lead to wrong biodiversity and stream quality assessments and molecular tools can greatly help resolving this problem. Here, we studied larvae of the caddisfly family Sericostomatidae in the Montseny mountains on the Iberian Peninsula. We expected to find cryptic species and that species would not occur in syntopy due to different ecological niches. We sampled 44 stream sites and sequenced 247 larval specimens for the barcoding region of the mitochondrial cytochrome c oxidase gene. A modeling approach was used to assess the bioclimatic preferences of the found species. Two molecular groups were identified. One could be assigned to Schizopelex furcifera and one to Sericostoma spp. We did find both taxa in syntopy in >50% of sampling sites and could show that the taxa prefer similar bioclimatic conditions. A reexamination of larval specimens showed that Sericostoma and Schizopelex larvae could not be unambiguously identified to the genus level. Overall, our results show the importance of including molecular tools into biodiversity assessments in order to correctly identify the species diversity of a region and to prevent wrong assessment results.

Molecular Biology

When is a bacterial "virulence factor" really virulent?

Bacterial traits that contribute to disease are termed virulence factors and there is much interest in therapeutic approaches that disrupt such traits. However, ecological theory predicts disease severity to be multifactorial and context dependent, which might complicate our efforts to identify the most generally important virulence factors. Here, we use meta-analysis to quantify disease outcomes associated with one well-studied virulence factor - pyoverdine, an iron-scavenging compound secreted by the opportunistic pathogen Pseudomonas aeruginosa. Consistent with ecological theory, we found that the effect of pyoverdine, albeit frequently contributing to disease, varied considerably across infection models. In many cases its effect was relatively minor, suggesting that pyoverdine is rarely essential for infections. Our work demonstrates the utility of meta-analysis as a tool to quantify variation and overall effects of purported virulence factors across different infection models. This standardised approach will help us to evaluate promising targets for anti-virulence approaches.

Microbiology

Defining the risk of Zika and chikungunya virus transmission in human population centers of the eastern United States

The recent spread of mosquito-transmitted viruses and associated disease to the Americas motivates a new, data-driven evaluation of risk in temperate population centers. Temperate regions are generally expected to pose low risk for significant mosquito-borne disease, however, the spread of the Asian tiger mosquito (Aedes albopictus) across densely populated urban areas has established a new landscape of risk. We use a model informed by field data to assess the conditions likely to facilitate local transmission of chikungunya and Zika viruses from an infected traveler to Ae. albopictus and then to other humans in USA cities with variable human densities and seasonality.\n\nMosquito-borne disease occurs when specific combinations of conditions maximize virus-to-mosquito and mosquito-to-human contact rates. We develop a mathematical model that captures the epidemiology and is informed by current data on vector ecology from urban sites. The model predicts that one of every two infectious travelers arriving at peak mosquito season could initiate local transmission and > 10% of the introductions could generate a disease outbreak of at least 100 people. Despite Ae. albopictus propensity for biting non-human vertebrates, we also demonstrate that local virus transmission and human outbreaks may occur when vectors feed from humans even just 40% of the time. This work demonstrates how a conditional series of non-average events can result in local arbovirus transmission and outbreaks of disease in humans, even in temperate cities.\n\nAuthor SummaryZika and chikungunya viruses are transmitted by Aedes mosquitoes, including Ae. albopictus, which is abundant in many temperate cities. While disease risk is lower in temperate regions where viral amplification cannot build across years, there is significant potential for localized disease outbreaks in urban populations. We use a model informed by field data to assess the conditions likely to facilitate local transmission of virus from an infected traveler to Ae. albopictus and then to other humans in USA cities with variable human densities and seasonality. The model predicts that one of every two infectious travelers arriving at peak mosquito season could initiate local transmission and > 10% of the introductions could generate a disease outbreak of >100 people.\n\nClassification: Ecology

Epidemiology

A massively parallel strategy for STR marker development, capture, and genotyping

Short tandem repeat (STRs or microsatellites) variants, are highly polymorphic markers that facilitate powerful, high-precision population genetic analyses. STRs are especially valuable in conservation and ecological genetic research, yielding detailed information on population structure and short-term demographic flux. However, STR marker development and analysis by conventional PCR-based methods imposes a workflow bottleneck and is suboptimal for noninvasive sampling strategies such as fecal DNA recovery. While massively parallel sequencing has not previously been leveraged for scalable, efficient STR recovery, here we present a pipeline for developing STR markers directly from high-throughput shotgun sequencing data without requiring a reference genome assembly, and a methodological approach for highly parallel recovery of enriched STR loci. We first employed our approach to design and capture a panel of 5,000 STR loci from a test group of diademed sifakas (Propithecus diadema, n=3), endangered Malagasy rainforest lemurs, and we report extremely efficient recovery of targeted loci--97.3-99.6% of STRs characterized with [≥]10x non-redundant coverage. Second, we tested our STR capture strategy on a P. diadema fecal DNA preparation, and report robust initial results and methodological suggestions for future implementations. In addition to STR targets, this approach also generates large, genome-wide single nucleotide polymorphism (SNP) panels from regions flanking the STR loci. Our method provides a cost-effective and highly scalable solution for rapid recovery of large STR and SNP datasets in any species without need for a reference genome, and can be used even with suboptimal DNA, which is more easily acquired in conservation and ecological genetic studies.\n\nData DepositionRaw sequencing data are available under Study Accession numbers SRP073167 (genomic shotgun data for Oberon and Tatiana) and SRP076225 (targeted re-sequencing data) from the NCBI Sequence Read Archive. BaitSTR software is available at Github (core BaitSTR programs: https://github.com/aakrosh/BaitSTR; BaitSTR_type.pl companion script for genotyping and block manipulation: https://github.com/lkistler/BaitSTR_type).

Genomics

Microbial Communities are Well Adapted to Disturbances in Energy Input

Although microbial systems are well-suited for studying concepts in ecological theory, little is known about how microbial communities respond to long-term periodic perturbations beyond diel oscillations. Taking advantage of an ongoing microcosm experiment, we studied how methanotrophic microbial communities adapted to disturbances in energy input over a 20 day cycle period. Sequencing of bacterial 16S rRNA genes together with quantification of microbial abundance and ecosystem function was used to explore the long-term dynamics (510 days) of methanotrophic communities under continuous versus cyclic chemical energy supply. We observed that microbial communities appear inherently well-adapted to disturbances in energy input and that changes in community structure in both treatments are more dependent on internal dynamics than on external forcing. Results also show that the rare biosphere is critical to seeding the internal community dynamics, perhaps due to cross-feeding or other strategies. We conclude that in our experimental system, endogenous feedbacks were more important than exogenous drivers in shaping the community dynamics over time, suggesting that ecosystems can maintain their function despite inherently unstable community dynamics.\n\nIMPORTANCEWithin the broader ecological context, biological communities are often viewed as stable and only experience succession or replacement when subject to external perturbations, such as changes in food availability or introduction of exotic species. Our findings indicate that microbial communities can exhibit strong internal dynamics that may be more important in shaping community succession than external drivers. Dynamic \"unstable\" communities may be important for ecosystem functional stability, with rare organisms playing an important role in community restructuring. Understanding the mechanisms responsible for internal community dynamics will certainly be required for understanding and manipulating microbiomes in both host-associated and natural ecosystems.

Microbiology

Genomic signatures of introgression at late stages of speciation in a malaria mosquito

Ongoing speciation in most African malaria vectors gives rise to cryptic populations, which differ remarkably in their behaviour, ecology and capacity to vector malaria parasites. Understanding the population structure and the drivers of genetic differentiation among mosquitoes is critical for effective disease control because heterogeneity within species contribute to variability in malaria cases and allow fractions of vector populations to escape control efforts. To examine the population structure and the potential impacts of recent large-scale control interventions, we have investigated the genomic patterns of differentiation in mosquitoes belonging to the Anopheles nili group, a large taxonomic group that diverged [~]3-Myr ago. Using 4343 single nucleotide polymorphisms (SNPs), we detected strong population structure characterized by high FST values between multiple divergent populations adapted to different habitats within the Central African rainforest. Delineating the cryptic species within the Anopheles nili group is challenging due to incongruence between morphology, ribosomal DNA and SNP markers consistent with incomplete lineage sorting and/or interspecific gene flow. A very high proportion of loci are fixed (FST = 1) within the genome of putative species, which suggests that ecological and/or reproductive barriers are maintained by strong selection on a substantial number of genes.

Evolutionary Biology