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Refactoring the Genetic Code for Increased Evolvability

The standard genetic code is robust to mutations and base-pairing errors during transcription and translation. Point mutations are most likely to be synonymous or preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best performing mutant requires a replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter single nucleotide per mutation event, due to the extreme rarity of adjacent mutations. In this theoretical study, we suggest a radical reordering of the genetic code that maximizes the mutagenic potential of single nucleotide replacements. We explore several possible genetic codes that allow a greater degree of accessibility to the mutational landscape and may result in a hyper-evolvable organism serving as an ideal platform for directed evolution experiments. We then conclude by evaluating potential applications for recoded organisms within the synthetic biology field.\n\nSignificance StatementThe conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene using common error-prone methods. Here we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transition to a larger pool of amino acids and with a greater degree of chemical differences, using a single nucleotide replacement within the codon, thus increasing evolvability both at the single gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation and fitness maximization.

bioengineering

Pleistocene climate change and the formation of regional species pools

This preprint has been reviewed and recommended by Peer Community In Evolutionary Biology (https://doi.org/10.24072/pci.evolbiol.100053). Despite the description of bioregions dates back from the origin of biogeography, the processes originating their associated species pools have been seldom studied. Ancient historical events are thought to play a fundamental role in configuring bioregions, but the effects of more recent events on these regional biotas are largely unknown. We use a network approach to identify regional and sub-regional faunas of European Carabus beetles, and analyse the effects of dispersal barriers, niche similarities and phylogenetic history on their configuration. We identify a transition zone matching the limit of the ice sheets at Last Glacial Maximum. While southern species pools are mostly separated by dispersal barriers, in the north species are mainly sorted by their environmental niches. Strikingly, most phylogenetic structuration of Carabus faunas occurred during the Pleistocene. Our results show how extreme recent historical events -such as Pleistocene climate cooling, rather than just deep-time evolutionary processes, can profoundly modify the composition and structure of geographic species pools.

ecology

Perspective: Genomic inference using diffusion models and the allele frequency spectrum

Evolutionary, biological, and demographic processes combine to shape the variation observed in populations. Understanding how these processes are expected to influence variation allows us to infer past demographic events and the nature of selection in human populations. Forward models such as the diffusion approximation provide a powerful tool for analyzing the distribution of allele frequencies in contemporary populations due to their computational tractability and model flexibility. Here, we discuss recent computational developments and their application to reconstructing human demographic history and patterns of selection at new mutations. We also reexamine how some classical assumptions that are still commonly used in inference studies fare when applied to modern data. We use whole-genome sequence data for 797 French Canadian individuals to examine the neutrality of synonymous sites. We find that selection can lead to strong biases in the inferred demography, mutation rate, and distributions of fitness effects. We use these distributions of fitness effects together with demographic and phenotype-fitness models to predict the relationship between effect size and allele frequency, and contrast those predictions to commonly used models in statistical genetics. Thus the simple evolutionary models investigated by Kimura and Ohta still provide important insight into modern genetic research.

genetics

A global perspective of codon usage

Codon usage in 2730 genomes is analyzed for evolutionary patterns in the usage of synonymous codons and amino acids across prokaryotic and eukaryotic taxa. We group genomes together that have similar amounts of intra-genomic bias in their codon usage, and then compare how usage of particular different codons is diversified across each genome group, and how that usage varies from group to group. Inter-genomic diversity of codon usage increases with intra-genomic usage bias, following a universal pattern. The frequencies of the different codons vary in robust mutual correlation, and the implied synonymous codon and amino acid usages drift together. This kind of correlation indicates that the variation of codon usage across organisms is chiefly a consequence of lateral DNA transfer among diverse organisms. The group of genomes with the greatest intra-genomic bias comprises two distinct subgroups, with each one restricting its codon usage to essentially one unique half of the genetic code table. These organisms include eubacteria and archaea thought to be closest to the hypothesized last universal common ancestor (LUCA). Their codon usages imply genetic diversity near the hypothesized base of the tree of life. There is a continuous evolutionary progression across taxa from the two extremely diversified usages toward balanced usage of different codons (as approached, e.g. in mammals). In that progression, codon frequency variations are correlated as expected from a blending of the two extreme codon usages seen in prokaryotes.\n\nAUTHOR SUMMARYThe redundancy intrinsic to the genetic code allows different amino acids to be encoded by up to six synonymous codons. Genomes of different organisms prefer different synonymous codons, a phenomenon known as codon usage bias. The phenomenon of codon usage bias is of fundamental interest for evolutionary biology, and is important in a variety of applied settings (e.g., transgene expression). The spectrum of codon usage biases seen in current organisms is commonly thought to have arisen by the combined actions of mutations and selective pressures. This view focuses on codon usage in specific genomes and the consequences of that usage for protein expression.\n\nHere we investigate an unresolved question of molecular genetics: are there global rules governing the usage of synonymous codons made by genomic DNA across organisms? To answer this question, we employed a data-driven approach to surveying 2730 species from all kingdoms of the tree of life in order to classify their codon usage. A first major result was that the large majority of these organisms use codons rather uniformly on the genome-wide scale, without giving preference to particular codons among possible synonymous alternatives. A second major result was that two compartments of codon usage seem to co-exist and to be expressed in different proportions by different organisms. As such, we investigate how individual different codons are used in different organisms from all taxa. Whereas codon usage is generally believed to be the evolutionary result of both mutations and natural selection, our results suggest a different perspective: the usage of different codons (and amino acids) by different organisms follows a superposition of two distinct patterns of usage. One distinction locates to the third base pair of all different codons, which in one pattern is U or A, and in the other pattern is G or C. This result has two major implications: (1) the variation of codon usage as seen across different organisms is best accounted for by lateral gene transfer among diverse organisms; (2) the organisms that are by protein homology grouped near the base of the tree of life comprise two genetically distinct lineages.\n\nWe find that, over evolutionary time, codon usages have converged from two distinct, non-overlapping usages (e.g., as evident in bacteria and archaea) to a near-uniform, balanced usage of synonymous codons (e.g., in mammals). This shows that the variations of codon (and amino acid) biases reveal a distinct evolutionary progression. We also find that codon usage in bacteria and archaea is most diverse between organisms thought to be closest to the hypothesized last universal common ancestor (LUCA). The dichotomy in codon (and amino acid usages) present near the origin of the current tree of life might provide information about the evolutionary development of the genetic code.

Genetics

Gene co-expression modules underlying polymorphic and monomorphic zooids in the colonial hydrozoan, Hydractinia symbiolongicarpus

Advances in sequencing technology have forced a quantitative revolution in Evolutionary Biology. One important feature of this renaissance is that comprehensive genomic resources can be obtained quickly for almost any taxon, thus speeding the development of new model organisms. Here we analyze 20 RNA-seq libraries from morphologically, sexually, and genetically distinct polyp types from the gonochoristic colonial hydrozoan, Hydractinia symbiolongicarpus (Cnidaria). Analyses of these data using Weighted Gene Co-expression Networks highlights deeply conserved genetic elements of animal spermatogenesis and demonstrate the utility of these methods in identifying modules of genes that correlate with different zooid types across various statistical contrasts. RNAseq data and analytical scripts described here are deposited in publicly available databases.

Developmental Biology

TESS: Bayesian inference of lineage diversification rates from (incompletely sampled) molecular phylogenies in R

SummaryMany fundamental questions in evolutionary biology entail estimating rates of lineage diversification (speciation - extinction). We develop a flexible Bayesian framework for specifying an effectively infinite array of diversification models--where rates are constant, vary continuously, or change episodically through time--and implement numerical methods to estimate parameters of these models from molecular phylogenies, even when species sampling is incomplete. Additionally we provide robust methods for comparing the relative and absolute fit of competing branching-process models to a given tree, thereby providing rigorous tests of biological hypotheses regarding patterns and processes of lineage diversification.\n\nAvailability and implementationthe source code for TESS is freely available at http://cran.r-project.org/web/packages/TESS/.\n\nContactSebastian.Hoehna@gmail.com

Bioinformatics

Shared generative rules of locomotor behavior in arthropods and vertebrates

The discovery of shared behavioral processes across phyla is an essential step in the establishment of a comparative study of behavior. We use immobility as an origin and reference for the measurement of locomotor behavior; speed, direction of walking and direction of facing as the three degrees of freedom shaping fly locomotor behavior; and cocaine as the parameter inducing a progressive transition in and out of immobility. In this way we expose and quantify the generative rules that shape part of fruit fly locomotor behavior, bringing about a gradual buildup of freedom during the transition from immobility to normal behavior and a precisely opposite narrowing down during the transition into immobility. During buildup the fly exhibits enhancement and then reduction to normal values of movement along each degree of freedom: first, body rotation in the horizontal plane, then path curvature and then speed of translation. Transition into immobility unfolds by narrowing down of the repertoire in the opposite sequential order, showing reciprocal relations during both buildup and narrowing down. The same generative rules apply to vertebrate locomotor behavior in a variety of contexts involving transition out and into immobility. Recent claims for deep homology between the arthropod central complex and the vertebrate basal ganglia provide an opportunity to examine whether the generative rules we discovered also share common descent. Neurochemical processes mediating the buildup of locomotor behavior in vertebrates could guide the search for equivalent processes in arthropods. The measurement methodology we use prompts the discovery of candidate behavioral homologies.\n\nSignificance StatementDo flies and mice share the same behavior? By defining immobility as an intrinsic reference point for locomotor behavior we show that the rules that generate the transition from immobility to full blown normal behavior, and from full blown behavior to immobility are shared by fruit flies and mice. These rules constitute a much desired aim of evolutionary biology: the discovery of behavioral homologies across distant phyla. The methodology we use facilitates the discovery of cross-phyletic behavioral homologies, shedding light on the problem of the evolution of behavior.

Animal Behavior and Cognition

Mega-analysis of 31,396 individuals from 6 countries uncovers strong gene-environment interaction for human fertility

Family and twin studies suggest that up to 50% of individual differences in human fertility within a population might be heritable. However, it remains unclear whether the genes associated with fertility outcomes such as number of children ever born (NEB) or age at first birth (AFB) are the same across geographical and historical environments. By not taking this into account, previous genetic studies implicitly assumed that the genetic effects are constant across time and space. We conduct a mega-analysis applying whole genome methods on 31,396 unrelated men and women from six Western countries. Across all individuals and environments, common single-nucleotide polymorphisms (SNPs) explained only ~4% of the variance in NEB and AFB. We then extend these models to test whether genetic effects are shared across different environments or unique to them. For individuals belonging to the same population and demographic cohort (born before or after the 20th century fertility decline), SNP-based heritability was almost five times higher at 22% for NEB and 19% for AFB. We also found no evidence suggesting that genetic effects on fertility are shared across time and space. Our findings imply that the environment strongly modifies genetic effects on the tempo and quantum of fertility, that currently ongoing natural selection is heterogeneous across environments, and that gene-environment interactions may partly account for missing heritability in fertility. Future research needs to combine efforts from genetic research and from the social sciences to better understand human fertility.\n\nAuthors SummaryFertility behavior - such as age at first birth and number of children - varies strongly across historical time and geographical space. Yet, family and twin studies, which suggest that up to 50% of individual differences in fertility are heritable, implicitly assume that the genes important for fertility are the same across both time and space. Using molecular genetic data (SNPs) from over 30,000 unrelated individuals from six different countries, we show that different genes influence fertility in different time periods and different countries, and that the genetic effects consistently related to fertility are presumably small. The fact that genetic effects on fertility appear not to be universal could have tremendous implications for research in the area of reproductive medicine, social science and evolutionary biology alike.

Genetics

Oligogenic Adaptation, Soft Sweeps, and Parallel Melanic Evolution in Drosophila melanogaster

Unraveling the genetic architecture of adaptive phenotypic divergence is a fundamental quest in evolutionary biology. In Drosophila melanogaster, high-altitude melanism has evolved in separate mountain ranges in sub-Saharan Africa, potentially as an adaptation to UV intensity. We investigated the genetic basis of this melanism in three populations using a new bulk segregant analysis mapping method. Although hundreds of genes are known to affect cuticular pigmentation in D. melanogaster, we identified only 19 distinct QTLs from 9 mapping crosses, with several QTL peaks being shared among two or all populations. Surprisingly, we did not find wide signals of genetic differentiation (Fst) between lightly and darkly pigmented populations at these QTLs, in spite of the pronounced phenotypic difference in pigmentation. Instead, we found small numbers of highly differentiated SNPs at the probable causative genes. A simulation analysis showed that these patterns of polymorphism are consistent with selection on standing genetic variation (leading to \"soft sweeps\"). Our results thus support a role for oligogenic selection on standing genetic variation in driving parallel ecological adaptation.

Genetics

Sexual dimorphism in the Drosophila metabolome increases throughout development

The expression of sexually dimorphic phenotypes from a shared genome between males and females is a longstanding puzzle in evolutionary biology. Increasingly, research has made use of transcriptomic technology to examine the molecular basis of sexual dimorphism through gene expression studies, but even this level of detail misses the metabolic processes that ultimately link gene expression with the whole organism phenotype. We use metabolic profiling in Drosophila melanogaster to complete this missing step, with a view to examining variation in male and female metabolic profiles, or metabolomes, throughout development. We show that the metabolome varies considerably throughout larval, pupal and adult stages. We also find significant sexual dimorphism in the metabolome, although only in pupae and adults, and the extent of dimorphism tends to increase throughout development. We compare this to transcriptomic data from the same population and find that the general pattern of increasing sex differences throughout development is mirrored in RNA expression. We discuss our results in terms of the usefulness of metabolic profiling in linking genotype and phenotype to more fully understand the basis of sexually dimorphic phenotypes.

Physiology

ENVIREM: An expanded set of bioclimatic and topographic variables increases flexibility and improves performance of ecological niche modeling

Species distribution modeling is a valuable tool with many applications across ecology and evolutionary biology. The selection of biologically meaningful environmental variables that determine relative habitat suitability is a crucial aspect of the modeling pipeline. The 19 bioclimatic variables from WorldClim are frequently employed, primarily because they are easily accessible and available globally for past, present and future climate scenarios. Yet, the availability of relatively few other comparable environmental datasets potentially limits our ability to select appropriate variables that will most successfully characterize a species distribution. We identified a set of 16 climatic and two topographic variables in the literature, which we call the O_SCPLOWENVIREMC_SCPLOW dataset, many of which are likely to have direct relevance to ecological or physiological processes determining species distributions. We generated this set of variables at the same resolutions as WorldClim, for the present, mid-Holocene, and Last Glacial Maximum (LGM). For 20 North American vertebrate species, we then assessed whether including the O_SCPLOWENVIREMC_SCPLOW variables led to improved species distribution models compared to models using only the existing WorldClim variables. We found that including the ENVIREM dataset in the pool of variables to select from led to substantial improvements in niche modeling performance in 17 out of 20 species. We also show that, when comparing models constructed with different environmental variables, differences in projected distributions were often greater in the LGM than in the present. These variables are worth consideration in species distribution modeling applications, especially as many of the variables have direct links to processes important for species ecology. We provide these variables for download at multiple resolutions and for several time periods at envirem.github.io. Furthermore, we have written the envirem R package to facilitate the generation of these variables from other input datasets.

Ecology

Despite reproductive interference, the net outcome of reproductive interactions among spider mite species is not necessarily costly

This preprint has been reviewed and recommended by Peer Community in Evolutionary Biology (http://dx.doi.org/10.24072/pci.evolbiol.100025). Reproductive interference is considered a strong ecological force, potentially leading to species exclusion. This supposes that the net effect of reproductive interactions is strongly negative for one of the species involved. Testing this requires a comprehensive analysis of interspecific reproductive interactions, accounting for the order and timing of mating events, and for their effects on either fertility or fecundity. To this aim, we measured reproductive interactions between a focal species, Tetranychus urticae, and an invasive (T.evansi) and a resident (T. ludeni) species, varying the mating sequence and interval, and measuring the effect of such crosses on fecundity and offspring sex ratio (a measure of fertility, as these species are haplodiploid). We found that mating with heterospecifics affected fecundity and sex ratio negatively, but also positively, depending on the species involved, and on the order and timing of mating events. Overall, the net effect of reproductive interactions was weak despite strong effects of particular events. In natural situations, the outcome of reproductive interactions will thus hinge upon the frequency of each event.

ecology

Individual Variation Of Natural D. melanogaster Associated Bacterial Communities

D. melanogaster has become an important model organism to study host-microbe interaction. However, we still know little about the natural microbial communities that are associated with D. melanogaster. Especially, information on inter-individual variation is still lacking because most studies so far have used pooled material from several flies. Here, we collected bacterial 16S rRNA gene community profiles from a set of 32 individuals from a single population and compare the variation to that of samples collected from different substrates and locations. While community differences were on average larger between samples collected from different substrates, there was still a surprising amount of variation of microbial communities between individual flies. The samples clustered into two groups suggesting that there are yet unknown factors that affect the composition of natural fly associated microbial communities and need research.\n\nImportanceD. melanogaster is an important model organism in evolutionary biology and also for the study of host-microbe interaction. In order to connect these to aspects of D. melanogaster biology, it is crucial to better understand the natural D. melanogaster microbiota because only the natural microbiota can affect the evolution of the host. We present, to our knowledge, the first data set that captures inter-individual variation of D. melanogaster associated bacterial communities. Clustering of communities into two larger groups suggests that there are important drivers of these communities that we do not understand yet suggesting in return that more research on the natural microbiota of D. melanogaster is needed.

microbiology

An experimental approach to understanding elevation limits in a montane terrestrial salamander, Plethodon montanus

Understanding the abiotic and biotic factors that determine the limits to species range is an essential goal in ecology, biogeography, evolutionary biology, and conservation biology. Moreover, predictions of shifts in species distributions under future changes in climate can be improved through understanding the spatial variation in survival, growth, and reproduction. A long-standing hypothesis postulates that, for Northern Hemisphere species, abiotic factors like temperature limit northern and/or higher elevation extents, while biotic factors like competition limit the southern and/or lower elevation range edges; though amphibians may not follow this general trend. Therefore, we combined environmental suitability models and a reciprocal transplant experiment across an elevational gradient to explore the role of the abiotic environment on the range limits of a montane salamander (Plethodon montanus). We first determined suitability of the abiotic environment for P. montanus, under current (1960 - 2000) and future (2050) climate scenarios. Second, we collected juveniles from each of three elevations and transplanted them within mesocosms such that each origin population was represented within each transplant location and vice-versa. We found that environmental suitability in 2050 decreased throughout the range compared to current predictions, especially at lower elevations. Additionally, we found that individuals starting body condition and transplant location were important predictors of survival, growth, and reproduction condition; importantly, individuals transplanted to low elevation had lower survival and growth rates compared to those moved to mid or high elevations. Our study provides experimental support that the abiotic environment limits the lower elevation distribution of P. montanus and, unfortunately, our results also paint a possible bleak future for this species and likely other montane terrestrial plethodontids. The abiotic environment, which will become increasingly limited under future changes in climate, was found to have more influence on survival and growth than population identity.

ecology

Genome-Wide SNP Discovery In Field And Laboratory Colonies Of Australian Plutella Species

Understanding dispersal and gene flow is an important focus of evolutionary biology, conservation biology and pest management. The diamondback moth, Plutella xylostella, is a worldwide pest of Brassica vegetable and oilseed cropping systems. This insect has high dispersal ability, which has important consequences for population dynamics and the potential spread of insecticide resistance genes. Population genetic studies of the diamondback moth have found little evidence of population structure, suggesting that frequent intermixing occurs within regions, however the patterns of local and regional dispersal remain to be identified. For this and many other pest species, understanding dispersal is crucial for developing integrated management tactics such as forecasting systems and insecticide resistance management plans. In recent years, next generation sequencing (NGS) methods have provided previously unparalleled resolution for population genetic studies in a wide range of species. Here, we assessed the potential of NGS-derived molecular markers to provide new insights about population structure in the diamondback moth. We use restriction-site-associated DNA sequencing (RAD-Seq) to discover hundreds to thousands of single nucleotide polymorphism (SNP) markers in nine field and laboratory-reared populations collected from Australia. Genotypic data from RAD-Seq markers identified a cryptic species, P. australiana, among individuals collected from a wild host, Diplotaxis sp., indicating strong divergence in the nuclear genomes of two Australian Plutella lineages. Significant genetic differentiation was detected among populations of P. xylostella used in our study, however this could be explained by reduced heterozogosity and genetic drift in laboratory-reared populations founded by relatively few individuals. This study demonstrates that RAD-Seq is a powerful method for generating SNP markers for population genetic studies in this species.

ecology

Genetic mapping of species differences via "in vitro crosses" in mouse embryonic stem cells

Discovering the genetic changes underlying species differences is a central goal in evolutionary genetics. However, hybrid crosses between species in mammals often suffer from hybrid sterility, greatly complicating genetic dissection of trait variation. Here we describe a simple, robust and transgene-free technique to make \"in vitro crosses\" in hybrid mouse embryonic stem cells by inducing random mitotic crossovers with the drug ML216, which inhibits Bloom syndrome (BLM). Starting with an interspecific hybrid (between Mus musculus and Mus spretus) embryonic stem cell line spanning 1.5 million years of divergence, we demonstrate the feasibility of mapping enzymatic differences across species within weeks and the possibility of re-deriving whole mice. Our work shows how in vitro crosses can overcome major bottlenecks like hybrid sterility in traditional mouse breeding to address fundamental questions in evolutionary biology.\n\nImpact StatementBy mixing hybrid mouse genomes in stem cells via mitotic recombination, genetic mapping and hybrid mosaic mice can be achieved in weeks, even across species barriers.

genetics

Trade-off between intra- and interannual scales in the evolution of aggressiveness in a local plant pathogen population

This preprint has been reviewed and recommended by Peer Community In Evolutionary Biology (http://dx.doi.org/10.24072/pci.evolbiol.100039). The efficiency of plant resistance to fungal pathogen populations is expected to decrease over time, due to its evolution with an increase in the frequency of virulent or highly aggressive strains. This dynamics may differ depending on the scale investigated (annual or pluriannual), particularly for annual crop pathogens with both sexual and asexual reproduction cycles. We assessed this time-scale effect, by comparing aggressiveness changes in a local Zymoseptoria tritici population over an eight-month cropping season and a six-year period of wheat monoculture. We collected two pairs of subpopulations to represent the annual and pluriannual scales: from leaf lesions at the beginning and end of a single annual epidemic, and from crop debris at the beginning and end of a six-year period. We assessed two aggressiveness traits - latent period and lesion size - on sympatric and allopatric host varieties. A trend toward decreased latent period concomitant with a significant loss of variability was established during the course of the annual epidemic, but not over the six-year period. Furthermore, a significant cultivar effect (sympatric vs. allopatric) on the average aggressiveness of the isolates revealed host adaptation, arguing that the observed patterns could result from selection. We thus provide an experimental body of evidence of an epidemiological trade-off between the intra- and inter-annual scales in the evolution of aggressiveness in a local plant pathogen population. More aggressive isolates were collected from upper leaves, on which disease severity is usually lower than on the lower part of the plants left in the field as crop debris after harvest. We suggest that these isolates play little role in sexual reproduction, due to an Allee effect (difficulty finding mates at low pathogen densities), particularly as the upper parts of the plant are removed from the field, explaining the lack of transmission of increases in aggressiveness between epidemics.

epidemiology

The genetic architecture of recurrent segregation distortion in Arabidopsis thaliana

The equal probability of transmission of alleles from either parent during sexual reproduction is a central tenet of genetics and evolutionary biology. Yet, there are many cases where this rule is violated. Such violations limit intraspecific gene flow and can facilitate the formation of genetic barriers, a first step in speciation. Biased transmission of alleles, or segregation distortion, can result from a number of biological processes including epistatic interactions between incompatible loci, gametic selection, and meiotic drive. Examples of these phenomena have been identified in many species, implying that they are universal, but comprehensive species-wide studies of segregation distortion are lacking. We have performed a species-wide screen for distorted allele frequencies in over 500 segregating populations of Arabidopsis thaliana using reduced-representation genome sequencing. Biased transmission of alleles was evident in up to a quarter of surveyed populations. Most populations exhibited distortion at only one genomic region, with some regions being repeatedly affected in multiple populations. Our results begin to elucidate the species-level architecture of biased transmission of genetic material in A. thaliana, and serve as a springboard for future studies into the basis of intraspecific genetic barriers.

plant biology