Temporal genetic stability in natural populations of the waterflea Daphnia magna in response to strong selection pressure

Studies monitoring changes in genetic diversity and composition through time allow a unique understanding of evolutionary dynamics and persistence of natural populations. However, such studies are often limited to species with short generation times that can be propagated in the laboratory or few exceptional cases in the wild. Species that produce dormant stages provide powerful models for the reconstruction of evolutionary dynamics in the natural environment. A remaining open question is to what extent dormant egg banks are an unbiased representation of populations and hence of the species’ evolutionary potential, especially in the presence of strong environmental selection. We address this key question using the water flea Daphnia magna, which produces dormant stages that accumulate in biological archives over time. We assess temporal genetic stability in three biological archives, previously used in resurrection ecology studies showing adaptive evolutionary responses to rapid environmental change. We show that neutral genetic diversity does not decline with the age of the population and it is maintained in the presence of strong selection. In addition, by comparing temporal genetic stability in hatched and unhatched populations from the same biological archive, we show that dormant egg banks can be consulted to obtain a reliable measure of genetic diversity over time, at least in the multidecadal time frame studied here. The stability of neutral genetic diversity through time is likely mediated by the buffering effect of the resting egg bank.     

Egg load dynamics and the risk of egg and time limitation experienced by an aphid parasitoid in the field

Insect parasitoids and herbivores must balance the risk of egg limitation and time limitation in order to maximize reproductive success. Egg and time limitation are mediated by oviposition and egg maturation rates as well as by starvation risk and other determinants of adult lifespan. Here, we assessed egg load and nutritional state in the soybean aphid parasitoid Binodoxys communis under field conditions to estimate its risk of becoming either egg‐ or time‐limited. The majority of female B. communis showed no signs of egg limitation. Experimental field manipulations of B. communis females suggested that an average of 4–8 eggs were matured per hour over the course of a day. Regardless, egg loads remained constant over the course of the day at approximately 80 eggs, suggesting that egg maturation compensates for oviposition. This is the first case of such “egg load buffering” documented for a parasitoid in the field. Despite this buffering, egg loads dropped slightly with increasing host (aphid) density. This suggests that egg limitation could occur at very high host densities as experienced in outbreak years in some locations in the Midwestern USA. Biochemical analyses of sugar profiles showed that parasitoids fed upon sugar in the field at a remarkably high rate. Time limitation through starvation thus seems to be very low and aphid honeydew is most likely a source of dietary sugar for these parasitoids. This latter supposition is supported by the fact that body sugar levels increase with host (aphid) density. Together, these results suggest that fecundity of B. communis benefits from both dynamic egg maturation strategies and sugar‐feeding.     

Population genetic structure of the bank vole Myodes glareolus within its glacial refugium in peninsular Italy

It is now well established that Southern European peninsulas have been major glacial refugia for temperate species during Pleistocene climatic oscillations. However, substantial environmental changes occurred also within these peninsulas throughout the Pleistocene, raising questions about the role and interplay of various microevolutionary processes in shaping patterns of intraspecific diversity within these areas. Here, we investigate the patterns of genetic variation in the bank vole Myodes glareolus within the Italian peninsula. By using a panel of 13 microsatellite loci, we found more intraspecific variation than expected based on previous assessments. Indeed, both Bayesian and ordination‐based clustering analyses of variation recovered five main geographic/genetic clusters along the peninsula, with three clusters geographically restricted to the southern portion of the peninsula. This clustering is supported by previous evidences of some morphological distinctiveness among these populations. This pattern can be explained by a refugia‐within‐refugia scenario, with the occurrence of multiple sub‐refugia for the bank vole within the Italian peninsula, likely promoted by the major palaeo‐environmental changes which affected forested habitats within this area during the Pleistocene. Moreover, our results support a scenario whereby the high levels of intraspecific diversity observed within major Pleistocene refugia are better explained by dynamic microevolutionary processes occurred within these areas, rather than by long‐term demographic stability of refugial population. Finally, the narrow and isolated distribution of some of the identified lineages suggests the need for future assessments of their conservation and taxonomic status.     

Population genetic analysis of a parasitic mycovirus to infer the invasion history of its fungal host

Hymenoscyphus fraxineus mitovirus 1 (HfMV1) occurs in the fungus Hymenoscyphus fraxineus, an introduced plant pathogen responsible for the devastating ash dieback epidemic in Europe. Here, we explored the prevalence and genetic structure of HfMV1 to elucidate the invasion history of both the virus and the fungal host. A total of 1298 H. fraxineus isolates (181 from Japan and 1117 from Europe) were screened for the presence of this RNA virus and 301 virus‐positive isolates subjected to partial sequence analysis of the viral RNA polymerase gene. Our results indicate a high mean prevalence (78.7%) of HfMV1 across European H. fraxineus isolates, which is supported by the observed high transmission rate (average 83.8%) of the mitovirus into sexual spores of its host. In accordance with an expected founder effect in the introduced population in Europe, only 1.1% of the Japanese isolates were tested virus positive. In Europe, HfMV1 shows low nucleotide diversity but a high number of haplotypes, which seem to be subject to strong purifying selection. Phylogenetic and clustering analysis detected two genetically distinct HfMV1 groups, both present throughout Europe. This pattern supports the hypothesis that only two (mitovirus‐carrying) H. fraxineus individuals were introduced into Europe as previously suggested from the bi‐allelic nature of the fungus. Moreover, our data points to reciprocal mating events between the two introduced individuals, which presumably initiated the ash dieback epidemic in Europe.     

Evidence for adaptation from standing genetic variation on an antimicrobial peptide gene in the mussel Mytilus edulis

Genome scans of population differentiation identify candidate loci for adaptation but provide little information on how selection has influenced the genetic structure of these loci. Following a genome scan, we investigated the nature of the selection responsible for the outlying differentiation observed between populations of the marine mussel Mytilus edulis at a leucine/arginine polymorphism (L31R) in the antimicrobial peptide MGD2. We analysed DNA sequence polymorphisms, allele frequencies and population differentiation of polymorphisms closely linked to L31R, and pairwise and third‐order linkage disequilibria. An outlying level of population differentiation was observed at L31R only, while no departure from panmixia was observed at linked loci surrounding L31R, as in most of the genome. Selection therefore seems to affect L31R directly. Three hypotheses can explain the lack of differentiation in the chromosomal region close to L31R: (i) hitchhiking has occurred but migration and recombination subsequently erased the signal, (ii) selection was weak enough and recombination strong enough to limit the hitchhiking effect to a very small chromosomal region or (iii) selection acted on a pre‐existing polymorphism (i.e. standing variation) at linkage equilibrium with its background. Linkage equilibrium was observed between L31R and linked polymorphisms in every population analysed, as expected under the three hypotheses. However, linkage disequilibrium was observed in some populations between pairs of loci located upstream and downstream to L31R, generating a complex pattern of third‐order linkage disequilibria which is best explained by the hypothesis of selection on a pre‐existing polymorphism. We hypothesise that selection could be either balanced, maintaining alleles at different frequencies depending on the pathogen community encountered locally by mussels, or intermittent, resulting in sporadic fluctuations in allele frequency.     

RhoJ modulates melanoma invasion by altering actin cytoskeletal dynamics

Rho family GTPases regulate diverse processes in human melanoma ranging from tumor formation to metastasis and chemoresistance. In this study, a combination of in vitro and in vivo approaches was utilized to determine whether RHOJ, a CDC42 homologue that regulates melanoma chemoresistance, also controls melanoma migration. Depletion or overexpression of RHOJ altered cellular morphology, implicating a role for RHOJ in modulating actin cytoskeletal dynamics. RHOJ depletion inhibited melanoma cell migration and invasion in vitro and melanoma tumor growth and lymphatic spread in mice. Molecular studies revealed that RHOJ alters actin cytoskeletal dynamics by inducing the phosphorylation of LIMK, cofilin, and p41‐ARC (ARP2/3 complex subunit) in a PAK1‐dependent manner in vitro and in tumor xenografts. Taken together, these observations identify RHOJ as a melanoma linchpin determinant that regulates both actin cytoskeletal dynamics and chemoresistance by activating PAK1.     

Inferences on population history of a seed chalcid wasp: invasion success despite a severe founder effect from an unexpected source population

Most invasive species established in Europe originate from either Asia or North America, but little is currently known about the potential of the Anatolian Peninsula (Asia Minor) and/or the Near East to constitute invasion sources. Mediterranean forests are generally fragile ecosystems that can be threatened by invasive organisms coming from different regions of the Mediterranean Basin, but for which historical data are difficult to gather and the phylogeographic patterns are still poorly understood for most terrestrial organisms. In this study, we characterized the genetic structure of Megastigmus schimitscheki, an invasive seed‐feeding insect species originating from the Near East, and elucidated its invasion route in South‐eastern France in the mid 1990s. To disentangle the evolutionary history of this introduction, we gathered samples from the main native regions (Taurus Mountains in Turkey, Lebanon and Cyprus) and from the invaded region that we genotyped using five microsatellite markers and for which we sequenced the mitochondrial Cytochrome Oxidase I gene. We applied a set of population genetic statistics and methods, including approximate Bayesian computation. We proposed a detailed phylogeographic pattern for the Near East populations, and we unambiguously showed that the French invasive populations originated from Cyprus, although the available historical data strongly suggested that Turkey could be the most plausible source area. Interestingly, we could show that the introduced populations were founded from an extremely restricted number of individuals that realized a host switch from Cedrus brevifolia to C. atlantica. Evolutionary hypotheses are discussed to account for this unlikely scenario.     

Daphnia diversity on the Tibetan Plateau measured by DNA taxonomy

Daphnia on the Tibetan Plateau has been little studied, and information on species diversity and biogeography is lacking. Here, we conducted a 4‐year survey using the barcoding fragment of the mitochondrial COI gene to determine the distribution and diversity of Daphnia species found across the Plateau. Our results show that species richness is higher than previously thought, with total described and provisional species number doubling from 5 to 10. Six of the taxonomic units recovered by DNA taxonomy agreed well with morphology, but DNA barcoding distinguished three clades each for the D. longispina (D. galeata, D. dentifera, and D. longispina) and D. pulex (D. pulex, D. cf. tenebrosa, and D. pulicaria) complexes. The sequence divergence between congeneric species varied within a large range, from 9.25% to 30.71%. The endemic D. tibetana was the most common and widespread species, occurring in 12 hyposaline to mesosaline lakes. The lineage of D. longispina is the first confirmed occurrence in west Tibet.     

Ecological and evolutionary factors of intraspecific variation in inducible defenses: Insights gained from Daphnia experiments

Phenotypic variation among individuals and species is a fundamental principle of natural selection. In this review, we focus on numerous experiments involving the model species Daphnia (Crustacea) and categorize the factors, especially secondary ones, affecting intraspecific variations in inducible defense. Primary factors, such as predator type and density, determine the degree to which inducible defense expresses and increases or decreases. Secondary factors, on the other hand, act together with primary factors to inducible defense or without primary factors on inducible defense. The secondary factors increase intraspecies variation in inducible defense, and thus, the level of adaptation of organisms varies within species. Future research will explore the potential for new secondary factors, as well as the relative importance between factors needs to be clarified.     

Population genetic structure,genetic diversity,and natural history of the South American species of Nothofagus subgenus Lophozonia (Nothofagaceae) inferred from nuclear microsatellite data

The effect of glaciation on the levels and patterns of genetic variation has been well studied in the Northern Hemisphere. However, although glaciation has undoubtedly shaped the genetic structure of plants in the Southern Hemisphere, fewer studies have characterized the effect, and almost none of them using microsatellites. Particularly, complex patterns of genetic structure might be expected in areas such as the Andes, where both latitudinal and altitudinal glacial advance and retreat have molded modern plant communities. We therefore studied the population genetics of three closely related, hybridizing species of Nothofagus (N. obliqua, N. alpina, and N. glauca, all of subgenus Lophozonia; Nothofagaceae) from Chile. To estimate population genetic parameters and infer the influence of the last ice age on the spatial and genetic distribution of these species, we examined and analyzed genetic variability at seven polymorphic microsatellite DNA loci in 640 individuals from 40 populations covering most of the ranges of these species in Chile. Populations showed no significant inbreeding and exhibited relatively high levels of genetic diversity (H_E = 0.502–0.662) and slight, but significant, genetic structure (R_ST = 8.7–16.0%). However, in N. obliqua, the small amount of genetic structure was spatially organized into three well‐defined latitudinal groups. Our data may also suggest some introgression of N. alpina genes into N. obliqua in the northern populations. These results allowed us to reconstruct the influence of the last ice age on the genetic structure of these species, suggesting several centers of genetic diversity for N. obliqua and N. alpina, in agreement with the multiple refugia hypothesis.     

Paleogenetic records of Daphnia pulicaria in two North American lakes reveal the impact of cultural eutrophication

Understanding the evolutionary consequences of the green revolution, particularly in wild populations, is an important frontier in contemporary biology. Because human impacts have occurred at varying magnitudes or time periods depending on the study ecosystem, evolutionary histories may vary considerably among populations. Paleogenetics in conjunction with paleolimnology enable us to associate microevolutionary dynamics with detailed information on environmental change. We used this approach to reconstruct changes in the temporal population genetic structure of the keystone zooplankton grazer, Daphnia pulicaria, using dormant eggs extracted from sediments in two Minnesota lakes (South Center, Hill). The extent of agriculture and human population density in the catchment of these lakes has differed markedly since European settlement in the late 19th century and is reflected in their environmental histories reconstructed here. The reconstructed environments of these two lakes differed strongly in terms of environmental stability and their associated patterns of Daphnia population structure. We detected long periods of stability in population structure and environmental conditions in South Center Lake that were followed by a dramatic temporal shift in population genetic structure after the onset of European settlement and industrialized agriculture in its watershed. In particular, we noted a 24.3‐fold increase in phosphorus (P) flux between pre‐European and modern sediment P accumulation rates (AR) in this lake. In contrast, no such shifts were detected in Hill Lake, where the watershed was not as impacted by European settlement and rates of change were less directional with a much smaller increase in sediment P AR (2.3‐fold). We identify direct and indirect effects of eutrophication proxies on genetic structure in these lake populations and demonstrate the power of using this approach in understanding the consequences of anthropogenic environmental change on natural populations throughout historic time periods.     

Whole genome amplification and sequencing of a Daphnia resting egg

Resting eggs banks are unique windows that allow us to directly observe shifts in population genetics, and phenotypes over time as natural populations evolve. Though a variety of planktonic organisms also produce resting stages, the keystone freshwater consumer, Daphnia, is a well‐known model for paleogenetics and resurrection ecology. Nevertheless, paleogenomic investigations are limited largely because resting eggs do not contain enough DNA for genomic sequencing. In fact, genomic studies even on extant populations include a laborious preparatory phase of batch culturing dozens of individuals to generate sufficient genomic DNA. Here, we furnish a protocol to generate whole genomes of single ephippial (resting) eggs and single daphniids. Whole genomes of single ephippial eggs and single adults were amplified using Qiagen REPLI‐g Single Cell kit reaction, followed by NEBNext Ultra DNA Library Prep Kit for library construction and Illumina sequencing. We compared the quality of the single‐egg and single‐individual amplified genomes to the standard batch genomic DNA extraction in the absence of genome amplification. At mean 20× depth, coverage was essentially identical for the amplified single individual relative to the unamplified batch extracted genome (>90% of the genome was covered and callable). Finally, while amplification resulted in the slight loss of heterozygosity for the amplified genomes, estimates were largely comparable and illustrate the utility and limitations of this approach in estimating population genetic parameters over long periods of time in natural populations of Daphnia and also other small species known to produce resting stages.     

Genetic diversity loss in a biodiversity hotspot: ancient DNA quantifies genetic decline and former connectivity in a critically endangered marsupial

The extent of genetic diversity loss and former connectivity between fragmented populations are often unknown factors when studying endangered species. While genetic techniques are commonly applied in extant populations to assess temporal and spatial demographic changes, it is no substitute for directly measuring past diversity using ancient DNA (aDNA). We analysed both mitochondrial DNA (mtDNA) and nuclear microsatellite loci from 64 historical fossil and skin samples of the critically endangered Western Australian woylie (Bettongia penicillata ogilbyi), and compared them with 231 (n = 152 for mtDNA) modern samples. In modern woylie populations 15 mitochondrial control region (CR) haplotypes were identified. Interestingly, mtDNA CR data from only 29 historical samples demonstrated 15 previously unknown haplotypes and detected an extinct divergent clade. Through modelling, we estimated the loss of CR mtDNA diversity to be between 46% and 91% and estimated this to have occurred in the past 2000–4000 years in association with a dramatic population decline. In addition, we obtained near‐complete 11‐loci microsatellite profiles from 21 historical samples. In agreement with the mtDNA data, a number of ‘new’ microsatellite alleles was only detected in the historical populations despite extensive modern sampling, indicating a nuclear genetic diversity loss >20%. Calculations of genetic diversity (heterozygosity and allelic rarefaction) showed that these were significantly higher in the past and that there was a high degree of gene flow across the woylie's historical range. These findings have an immediate impact on how the extant populations are managed and we recommend the implementation of an assisted migration programme to prevent further loss of genetic diversity. Our study demonstrates the value of integrating aDNA data into current‐day conservation strategies.     

Inbreeding and adaptive plasticity: an experimental analysis on predator‐induced responses in the water flea Daphnia

Several studies have emphasized that inbreeding depression (ID) is enhanced under stressful conditions. Additionally, one might imagine a loss of adaptively plastic responses which may further contribute to a reduction in fitness under environmental stress. Here, we quantified ID in inbred families of the cyclical parthenogen Daphnia magna in the absence and presence of fish predation risk. We test whether predator stress affects the degree of ID and if inbred families have a reduced capacity to respond to predator stress by adaptive phenotypic plasticity. We obtained two inbred families through clonal selfing within clones isolated from a fish pond. After mild purging under standardized conditions, we compared life history traits and adaptive plasticity between inbred and outbred lineages (directly hatched from the natural dormant egg bank of the same pond). Initial purging of lineages under standardized conditions differed among inbred families and exceeded that in outbreds. The least purged inbred family exhibited strong ID for most life history traits. Predator‐induced stress hardly affected the severity of ID, but the degree to which the capacity for adaptive phenotypic plasticity was retained varied strongly among the inbred families. The least purged family overall lacked the capacity for adaptive phenotypic plasticity, whereas the family that suffered only mild purging exhibited a potential for adaptive plasticity that was comparable to the outbred population. We thus found that inbred offspring may retain the capacity to respond to the presence of fish by adaptive phenotypic plasticity, but this strongly depends on the parental clone engaging in selfing.     

Egg color variation,but not egg rejection behavior,changes in a cuckoo host breeding in the absence of brood parasitism

Interactions between parasitic cuckoos and their songbird hosts form a classical reciprocal “arms race,” and are an excellent model for understanding the process of coevolution. Changes in host egg coloration via the evolution of interclutch variation in egg color or intraclutch consistency in egg color are hypothesized counter adaptations that facilitate egg recognition and thus limit brood parasitism. Whether these antiparasitism strategies are maintained when the selective pressure of parasitism is relaxed remains debated. However, introduced species provide unique opportunities for testing the direction and extent of natural selection on phenotypic trait maintenance and variation. Here, we investigated egg rejection behavior and egg color polymorphism in the red‐billed leiothrix (Leiothrix lutea), a common cuckoo (Cuculus canorus) host, in a population introduced to Hawaii 100 years ago (breeding without cuckoos) and a native population in China (breeding with cuckoos). We found that egg rejection ability was equally strong in both the native and the introduced populations, but levels of interclutch variation and intraclutch consistency in egg color in the native population were higher than in the introduced population. This suggests that egg rejection behavior in hosts can be maintained in the absence of brood parasitism and that egg appearance is maintained by natural selection as a counter adaptation to brood parasitism. This study provides rare evidence that host antiparasitism strategies can change under parasite‐relaxed conditions and reduced selection pressure.     

Predators can influence the host‐parasite dynamics of their prey via nonconsumptive effects

Ecological communities are partly structured by indirect interactions, where one species can indirectly affect another by altering its interactions with a third species. In the absence of direct predation, nonconsumptive effects of predators on prey have important implications for subsequent community interactions. To better understand these interactions, we used a Daphnia‐parasite‐predator cue system to evaluate if predation risk affects Daphnia responses to a parasite. We investigated the effects of predator cues on two aspects of host–parasite interactions (susceptibility to infection and infection intensity), and whether or not these effects differed between sexes. Our results show that changes in response to predator cues caused an increase in the prevalence and intensity of parasite infections in female predator‐exposed Daphnia. Importantly, the magnitude of infection risk depended on how long Daphnia were exposed to the cues. Additionally, heavily infected Daphnia that were constantly exposed to cues produced relatively more offspring. While males were ~5× less likely to become infected compared to females, we were unable to detect effects of predator cues on male Daphnia–parasite interactions. In sum, predators, prey, and their parasites can form complex subnetworks in food webs, necessitating a nuanced understanding of how nonconsumptive effects may mediate these interactions.     

Population genetic structure and adaptive differentiation of iron walnut Juglans regia subsp. sigillata in southwestern China

Southwestern (SW) China is an area of active tectonism and erosion, yielding a dynamic, deeply eroded landscape that influences the genetic structure of the resident populations of plants and animals. Iron walnut (Juglans regia subsp. sigillata) is a deciduous tree species endemic to this region of China and cultivated there for its edible nuts. We sampled 36 iron walnut populations from locations throughout the species' range in SW China and genotyped a total of 765 individuals at five chloroplast DNA regions and 22 nuclear microsatellite loci. Species distribution models were produced to predict the evolution and historical biogeography of iron walnut and to estimate the impacts of climate oscillations and orographic environments on the species' demography. Our results indicated that J. regia subsp. sigillata had relatively low genetic diversity, high interpopulation genetic differentiation, and asymmetric interpopulation gene flow. Based on DIYABC analysis, we identified two lineages of J. sigillata in southwestern China. The lineages (subpopulations) diverge during the last glacial period (~1.34 Ma). Southwestern China was a glacial refuge during the last glacial period, but increasingly colder and arid climates might have fostered the fragmentation of J. regia subsp. sigillata within this refugium. Finally, we found that recent habitat fragmentation has led to a reduction in population connectivity and increased genetic differentiation by genetic drift in isolated populations. Our results support a conclusion that geological and climatic factors since the Miocene triggered the differentiation, evolutionary origin, and range shifts of J. sigillata in the studied region.