Hybridization and the Origin of Contagious Asexuality in Daphnia pulex.

Hybridization plays a potentially important role in the origin of obligate parthenogenesis (OP) in many organisms. However, it remains controversial whether hybridization directly triggers the transition from sexual reproduction to obligate asexuality or a hybrid genetic background enables asexual species to persist. Furthermore, we know little about the specific genetic elements from the divergent, yet still hybridizing lineages responsible for this transition and how these elements are further spread to create other OP lineages. In this study, we address these questions in Daphnia pulex, where cyclically parthenogenetic (CP) and OP lineages coexist. Ancestry estimates and whole-genome association mapping using 32 OP isolates suggest that a complex hybridization history between the parental species D. pulex and D. pulicaria is responsible for the introgression of a set of 647 D. pulicaria single nucleotide polymorphism alleles that show perfect association with OP. Crossing experiments using males of OP lineages and females of CP lineages strongly support a polygenic basis for OP. Single-sperm analyses show that although normal meiotic recombination occurs in the production of haploid sperm by males of OP lineages, a significant proportion of such sperm are polyploid, suggesting that the spread of asexual elements through these males (i.e., contagious asexuality) is much less efficient than previously envisioned. Although the current Daphnia genome annotation does not provide mechanistic insight into the nature of the asexuality-associated alleles, these alleles should be considered as candidates for future investigations on the genetic underpinnings of OP.

Population Genomics of Daphnia pulex.

Using data from 83 isolates from a single population, the population genomics of the microcrustacean Daphnia pulex are described and compared to current knowledge for the only other well-studied invertebrate, Drosophila melanogaster These two species are quite similar with respect to effective population sizes and mutation rates, although some features of recombination appear to be different, with linkage disequilibrium being elevated at short ([Formula: see text] bp) distances in D. melanogaster and at long distances in D. pulex The study population adheres closely to the expectations under Hardy-Weinberg equilibrium, and reflects a past population history of no more than a twofold range of variation in effective population size. Fourfold redundant silent sites and a restricted region of intronic sites appear to evolve in a nearly neutral fashion, providing a powerful tool for population genetic analyses. Amino acid replacement sites are predominantly under strong purifying selection, as are a large fraction of sites in UTRs and intergenic regions, but the majority of SNPs at such sites that rise to frequencies [Formula: see text] appear to evolve in a nearly neutral fashion. All forms of genomic sites (including replacement sites within codons, and intergenic and UTR regions) appear to be experiencing an [Formula: see text] higher level of selection scaled to the power of drift in D. melanogaster, but this may in part be a consequence of recent demographic changes. These results establish D. pulex as an excellent system for future work on the evolutionary genomics of natural populations.

Estimating Seven Coefficients of Pairwise Relatedness Using Population-Genomic Data.

Population structure can be described by genotypic-correlation coefficients between groups of individuals, the most basic of which are the pairwise relatedness coefficients between any two individuals. There are nine pairwise relatedness coefficients in the most general model, and we show that these can be reduced to seven coefficients for biallelic loci. Although all nine coefficients can be estimated from pedigrees, six coefficients have been beyond empirical reach. We provide a numerical optimization procedure that estimates all seven reduced coefficients from population-genomic data. Simulations show that the procedure is nearly unbiased, even at 3× coverage, and errors in five of the seven coefficients are statistically uncorrelated. The remaining two coefficients have a negative correlation of errors, but their sum provides an unbiased assessment of the overall correlation of heterozygosity between two individuals. Application of these new methods to four populations of the freshwater crustacean Daphnia pulex reveal the occurrence of half siblings in our samples, as well as a number of identical individuals that are likely obligately asexual clone mates. Statistically significant negative estimates of these pairwise relatedness coefficients, including inbreeding coefficients that were typically negative, underscore the difficulties that arise when interpreting genotypic correlations as estimations of the probability that alleles are identical by descent.

A New Reference Genome Assembly for the Microcrustacean Daphnia pulex.

Comparing genomes of closely related genotypes from populations with distinct demographic histories can help reveal the impact of effective population size on genome evolution. For this purpose, we present a high quality genome assembly of Daphnia pulex (PA42), and compare this with the first sequenced genome of this species (TCO), which was derived from an isolate from a population with >90% reduction in nucleotide diversity. PA42 has numerous similarities to TCO at the gene level, with an average amino acid sequence identity of 98.8 and >60% of orthologous proteins identical. Nonetheless, there is a highly elevated number of genes in the TCO genome annotation, with ∼7000 excess genes appearing to be false positives. This view is supported by the high GC content, lack of introns, and short length of these suspicious gene annotations. Consistent with the view that reduced effective population size can facilitate the accumulation of slightly deleterious genomic features, we observe more proliferation of transposable elements (TEs) and a higher frequency of gained introns in the TCO genome.

Promoter Architecture and Sex-Specific Gene Expression in Daphnia pulex.

Large-scale transcription start site (TSS) profiling produces a high-resolution, quantitative picture of transcription initiation and core promoter locations within a genome. However, application of TSS profiling to date has largely been restricted to a small set of prominent model systems. We sought to characterize the cis-regulatory landscape of the water flea Daphnia pulex, an emerging model arthropod that reproduces both asexually (via parthenogenesis) and sexually (via meiosis). We performed Cap Analysis of Gene Expression (CAGE) with RNA isolated from D. pulex within three developmental states: sexual females, asexual females, and males. Identified TSSs were utilized to generate a "Daphnia Promoter Atlas," i.e., a catalog of active promoters across the surveyed states. Analysis of the distribution of promoters revealed evidence for widespread alternative promoter usage in D. pulex, in addition to a prominent fraction of compactly-arranged promoters in divergent orientations. We carried out de novo motif discovery using CAGE-defined TSSs and identified eight candidate core promoter motifs; this collection includes canonical promoter elements (e.g., TATA and Initiator) in addition to others lacking obvious orthologs. A comparison of promoter activities found evidence for considerable state-specific differential gene expression between states. Our work represents the first global definition of transcription initiation and promoter architecture in crustaceans. The Daphnia Promoter Atlas presented here provides a valuable resource for comparative study of cis-regulatory regions in metazoans, as well as for investigations into the circuitries that underpin meiosis and parthenogenesis.

A Male-Specific Genetic Map of the Microcrustacean Daphnia pulex Based on Single-Sperm Whole-Genome Sequencing.

Genetic linkage maps are critical for assembling draft genomes to a meaningful chromosome level and for deciphering the genomic underpinnings of biological traits. The estimates of recombination rates derived from genetic maps also play an important role in understanding multiple aspects of genomic evolution such as nucleotide substitution patterns and accumulation of deleterious mutations. In this study, we developed a high-throughput experimental approach that combines fluorescence-activated cell sorting, whole-genome amplification, and short-read sequencing to construct a genetic map using single-sperm cells. Furthermore, a computational algorithm was developed to analyze single-sperm whole-genome sequencing data for map construction. These methods allowed us to rapidly build a male-specific genetic map for the freshwater microcrustacean Daphnia pulex, which shows significant improvements compared to a previous map. With a total of mapped 1672 haplotype blocks and an average intermarker distance of 0.87 cM, this map spans a total genetic distance of 1451 Kosambi cM and comprises 90% of the resolved regions in the current Daphnia reference assembly. The map also reveals the mistaken mapping of seven scaffolds in the reference assembly onto chromosome II by a previous microsatellite map based on F2 crosses. Our approach can be easily applied to many other organisms and holds great promise for unveiling the intragenomic and intraspecific variation in the recombination rates.

CHAOBORUS PREDATION AND LIFE-HISTORY EVOLUTION IN DAPHNIA PULEX: TEMPORAL PATTERN OF POPULATION DIVERSITY, FITNESS, AND MEAN LIFE HISTORY.

The effect of predation by the aquatic dipteran larva Chaoborus americanus on genetic diversity and life-history evolution in the cladoceran Daphnia pulex was investigated in large replicate laboratory populations. Instantaneous daily loss rates of clonal diversity and genetic variance for fitness indicate that 93-99% of initial genetic diversity can be removed from populations during the 8-12 generations of clonal reproduction that occur each year in natural populations. In the absence of predation, the principal evolved changes in mean population life history were smaller immature body size and increased and earlier fecundity. In the presence of size-selective Chaoborus predation, populations evolved toward larger body size and increased and earlier reproduction. The difference between these two trajectories is an estimate of the direct additive effect of Chaoborus predation. This effect was manifested as evolution toward larger body size with a trend toward earlier and increased reproduction.

THE COVARIANCE STRUCTURE OF LIFE-HISTORY CHARACTERS IN DAPHNIA PULEX.

The genetic covariance structure for life-history characters in two populations of cyclically parthenogenetic Daphnia pulex indicates considerable positive correlation among important fitness components, apparently at odds with the expectation if antagonistic pleiotropy is the dominant cause of the maintanence of genetic variation. Although there is no genetic correlation between offspring size and offspring number, present growth and present reproduction are both strongly positively correlated genetically with future reproduction, and early maturity is genetically correlated with larger clutch size. Although the ubiquity of antagonistic pleiotropy has been recently questioned, there are peculiarities of cyclical parthenogenesis that could lead to positive life-history covariance even when negative covariance would be expected in a similar sexual species. These include the influence of nonadditive gene action on evolution in clonally reproducing organisms, and the periodic release of hidden genetic variance within populations of cyclical parthenogens. Examination of matrix similarity, using the bootstrap for distribution-free hypothesis testing, reveals no evidence to suggest that the genetic covariance matrices differ between the populations. However, there is considerable evidence that the phenotypic and environmental covariance matrices differ between populations. These results indicate approximate stability of the genetic covariance matrix within species, an important assumption of many phenotypic evolution models, but should caution against the use of phenotypic in place of genetic covariance matrices.

THE DISTRIBUTION OF LIFE-HISTORY VARIATION IN THE DAPHNIA PULEX COMPLEX.

In the midwestern United States the Daphnia pulex complex consists of a mosaic of sexual and asexual populations, providing a useful model system for studying the evolutionary forces underlying the maintenance of sex. One asexual and two sexual populations were surveyed for genetic variation for isozymes, mitochondrial DNA, and life-history characters. While the sexual populations exhibited substantial levels of genetic variance for fitness characters, no variation was detected in the asexual population at any level. However, a parallel survey among asexual clones derived from other ponds revealed large amounts of quantitative variation among clones, even among those with the same molecular profile. As a group, the asexuals are more variable for life histories than are the sexual populations. The molecular data indicate a relatively recent origin for the extant asexual D. pulex. The polyphyletic origin of these clones, combined with their microevolutionary potential, provides an explanation for their broad geographic distribution. The distribution of sex in the complex cannot be explained with the standard models that assume an invariant asexual population in reproductive isolation from the parental species. Although the frequency of asexuality may be driven by the spread of a sex-limited meiosis suppressor through sexual populations, the complete displacement of sexuality may be prevented by ecological distinctions between the two classes of individuals. On average, the asexuals are larger but produce smaller clutches than the sexuals.

THE DETERMINATION OF GENETIC COVARIANCES AND PREDICTION OF EVOLUTIONARY TRAJECTORIES BASED ON A GENETIC CORRELATION MATRIX.

There is much interest in measuring selection, quantifying evolutionary constraints, and predicting evolutionary trajectories in natural populations. For these studies, genetic (co)variances among fitness traits play a central role. We explore the conditions that determine the sign of genetic covariances and demonstrate a critical role of selection in shaping genetic covariances. In addition, we show that genetic covariance matrices rather than genetic correlation matrices should be characterized and studied in order to infer genetic basis of population differentiation and/or to predict evolutionary trajectories.

THE QUANTITATIVE AND MOLECULAR GENETIC ARCHITECTURE OF A SUBDIVIDED SPECIES.

In an effort to elucidate the evolutionary mechanisms that determine the genetic architecture of a species, we have analyzed 17 populations of the microcrustacean Daphnia pulex for levels of genetic variation at the level of life-history characters and molecular markers in the nuclear and mitochondrial genomes. This species is highly subdivided, with approximately 30% of the variation for nuclear molecular markers and 50% of the variation for mitochondrial markers being distributed among populations. The average level of genetic subdivision for quantitative traits is essentially the same as that for nuclear markers, which superficially suggests that the life-history characters are diverging at the neutral rate. However, the existence of a strong correlation between the levels of population subdivision and broadsense heritabilities of individual traits argues against this interpretation, suggesting instead that the among-population divergence of some quantitative traits (most notably body size) is being driven by local adaptation to different environments. The fact that the mean phenotypes of the individual populations are also strongly correlated with local levels of homozygosity indicates that variation in local inbreeding plays a role in population differentiation. Rather than being a passive consequence of local founder effects, levels of homozygosity may be selected for directly for their effects on the phenotype (adaptive inbreeding depression). There is no relationship between the levels of variation within populations for molecular markers and quantitative characters, and this is explained by the fact that the average standing genetic variation for life-history characters in this species is equivalent to only 33 generations of variation generated by mutation.