The effect of diapause on the genetic structure of Daphnia magna populations

For intermittent Daphnia magna populations embryonic diapause following bisexual reproduction is the means of surviving in harsh environmental conditions. Another advantage of bisexuality is genetic recombination which produces a variety of genotypes. Crowding and short-day photo-periods promote bisexual reproduction, but photoperiod, at least, can not be an obligatory factor in D. magna since some bisexual females have been found also during the periods of long daylength. Because the catastrophe dates vary considerably, the most advantageous reproductive strategy must be one with some flexibility in reacting to irregular catastrophes by producing ephippial resting eggs whenever the probability of a catastrophe exceeds a certain threshold value. Extensive genetic changes occurring during diapause suggest that there are considerable genetic differences in ephippial egg production and egg survival. Genetic drift alone can not account for the great genetic changes observed, although in populations reacting slowly to changing conditions the number of diapausing ephippia can be relatively low. If the ephippia hatch over a long period of time, they can act as a stabilizing mechanism for genotype frequencies.     

Salinity tolerance in <Emphasis Type="Italic">Daphnia magna</Emphasis>: characteristics of genotypes hatching from mixed sediments

The hatching of diapausing eggs is a means of temporal dispersal that can provide populations with genotypes adapted to different environments. In a salinity-variable shallow lake, we predicted that the mixing of different age-classes of eggs in the sediment may yield genotypes with different salinity optima. The alternative would be the absence of local adaptation and the presence of a homogenous population of salt-tolerant genotypes with high phenotypic plasticity. We tested these alternatives by isolating Daphnia magna resting eggs from different sediment depths, exposing them to hatching cues at different salinity levels and measuring the performance of hatched individuals. Results revealed a homogeneous sediment with generally broad-tolerance genotypes and some genotypes with low salt tolerance, which supports the second hypothesis. However, the disturbed character of the sediment hampered historical reconstruction. The absence of local adaptation in the diapausing egg bank may be the result of various scenarios in the response of D. magna populations to severe salinity changes in the lake.     

Status of Daphnia Resting Egg Banks in Bohemian Forest Lakes Affected by Acidification

We studied the egg banks of Daphnia gr. longispina in four acidified lakes in the Bohemian Forest (Czech Republic, Europe). Daphnia had become extinct in three of the studied lakes due to anthropogenic acidification and we assessed the possibility of autochthonous recovery of the population after recent amelioration of the lake environment. We determined the ephippia distribution in eleven cores 10–30 cm long, and the state of Daphnia resting eggs in over 13 000 ephippia from eight cores. Apparently well-preserved eggs were used for hatching experiments and for DNA amplification. Vertical profiles of ephippia densities in several cores did not agree with historical data on the Daphnia presence in the lakes, as the sediment had been repeatedly disturbed by human activities in the past. Ephippia are present near the surface of the sediment in all lakes, and they might therefore receive hatching cues. We were not able, however, to prove that viable eggs are present in the ephippia banks of the lakes. The percentage of the well-preserved eggs in all but one core was below 1%. In addition, no egg hatched in the hatching experiments and we were not able to amplify DNA from the preserved eggs of lakes where Daphnia is extinct, although amplification from relatively young eggs from the fourth lake was successful in 60% of the cases. We conclude that the recovery of Daphnia populations in studied lakes from autochthonous sources is unlikely.     

Effects of algal food quality on sexual reproduction of Daphnia magna

The objective of our study was to investigate sexual reproduction of Daphnia magna associated with mating behaviors and hatching rates, according to different algal food sources. Since a diatom is known to contain more abundant long‐chain poly unsaturated fatty acids (PUFAs), we hypothesized that the diatom‐consuming D. magna would exhibit more successful reproduction rates. Upon the hypothesis, we designed three experiments using two algal species, a green alga (Chlorella vulgaris) and a diatom (Stephanodiscus hantzschii). From the results, we found that the mating frequency and copulation duration increased in the treatment with S. hantzschii, resulting in a significant increase of hatching rates of resting eggs. In the other two repetitive mating strategies (e.g., one female vs. multiple males, and one male vs. multiple females), we found that the hatching rates of resting eggs were greater in the S. hantzschii treatment. In addition to the mating strategy, male body size significantly increased in the diatom treatment, hence average diameter of penis was also statistically different among the treatments (greater diameter in the S. hantzschii treatment). To examine the effect of algal food quality, we estimated quantity of fatty acids in the two algal species. Our result showed that S. hantzschii had a higher proportion of long‐chain PUFAs than C. vulgaris. Furthermore, a stable isotope analysis revealed that carbon and nitrogen originated from S. hantzschii were more assimilated to D. magna. In summary, our study manifested that diatom consumption of D. magna leads to more successful sexual reproduction. We then discussed how the diatom consumption of zooplankton influences food web dynamics in a freshwater ecosystem.     

Body length, size of resting eggs and identification of the ephippia of Ceriodaphnia megalops and Ceriodaphnia pulchella

The ephippia of Ceriodaphnia megalops and Ceriodaphnia pulchella are described. Their ephippia may be identified by their size and shape, supplemented by measurement of the length of the resting egg. The ephippia are shed free in the water and float by means of a special area of gas-filled tissue. Each ephippium contained one ellipsoid resting egg. The length, width and shape of the resting eggs of both species were investigated, and comparisons made with published data of their parthenogenetic eggs. For both species the volume of a resting egg was virtually the same as the volume of a parthenogenetic egg, but the resting egg was more elongated. The shape of resting eggs was linearly related to egg volume on logarithmic axes in a manner similar to that reported for the parthenogenetic eggs of these two species. The linear relationship between total parthenogenetic egg volume and body length reported for these two species did not apply to their resting eggs.     

High‐resolution melting analysis: a genotyping tool for population studies on Daphnia

Determining genetic variation at the DNA level within and between natural populations is important for understanding the role of natural selection on phenotypic traits, but many techniques of screening for genetic variation are either cost intensive, not sensitive enough or too labour‐ and time‐consuming. Here, we demonstrate high‐resolution melting analysis (HRMA) as a cost‐effective and powerful tool for screening variable target genes in natural populations. HRMA is based on monitoring the melting of PCR amplicons. Owing to saturating concentrations of a dye that binds at high concentrations to double‐stranded DNA, it is possible to genotype high numbers of samples rapidly and accurately. We analysed digestive trypsins of two Daphnia magna populations as an application example for HRMA. One population originated from a pond containing toxic cyanobacteria that possibly produce protease inhibitors and the other from a pond without such cyanobacteria. The hypothesis was that D. magna clones from ponds with cyanobacteria have undergone selection by these inhibitors, which has led to different trypsin alleles. We first sequenced pooled genomic PCR products of trypsins from both populations to identify variable DNA sequences of active trypsins. Second, we screened variable DNA sequences of each D. magna clone from both populations for single nucleotide polymorphisms via HRMA. The HRMA results revealed that both populations exhibited phenotypic differences in the analysed trypsins. Our results indicate that HRMA is a powerful genotyping tool for studying the variation of target genes in response to selection within and between natural Daphnia populations.     

Overcrowding,food and phosphorus limitation effects on ephipphia production and population dynamics in the invasive species <Emphasis Type="Italic">Daphnia lumholtzi</Emphasis>

Daphnia lumholtzi has been very successful in colonizing North America since its appearance in Texas in 1990. Although previous studies have sought to link its success as an invasive species with various aspects of its population biology, there is little experimental data linking the invasion success of D. lumholtzi with its autecology, specifically its reproduction strategy. In this study we sought to link food quality and quantity to diapause in D. lumholtzi through a variation in phosphorus (P) content of algae, food quantity, and light level. We also assessed the effect of Daphnia peak population densities on reproductive rates and production of resting eggs. We found that when food is abundant, per capita ephippia production may be limited by P, but under food limitation conditions, there is no significant effect of food quality on ephippia production. Our results suggest that a combination of food quality/quantity and population density may work together to induce the production of resting eggs in this invasive species. Handling editor: S. Declerk     

A limit on the extent to which increased egg size can compensate for a poor postnatal environment revealed experimentally in the burying beetle,Nicrophorus vespilloides

It is often assumed that there is a positive relationship between egg size and offspring fitness. However, recent studies have suggested that egg size has a greater effect on offspring fitness in low‐quality environments than in high‐quality environments. Such observations suggest that mothers may compensate for poor posthatching environments by increasing egg size. In this paper we test whether there is a limit on the extent to which increased egg size can compensate for the removal of posthatching parental care in the burying beetle, Nicrophorus vespilloides. Previous experiments with N. vespilloides suggest that an increased egg size can compensate for a relatively poor environment after hatching. Here, we phenotypically engineered female N. vespilloides to produce large or small eggs by varying the amount of time they were allowed to feed on the carcass as larvae. We then tested whether differences between these groups in egg size translated into differences in larval performance in a harsh postnatal environment that excluded parental care. We found that females engineered to produce large eggs did not have higher breeding success, and nor did they produce larger larvae than females engineered to produce small eggs. These results suggest that there is a limit on the extent to which increased maternal investment in egg size can compensate for a poor posthatching environment. We discuss the implication of our results for a recent study showing that experimental N. vespilloides populations can adapt rapidly to the absence of posthatching parental care.     

High genetic variation in resting‐stage production in a metapopulation: Is there evidence for local adaptation?

Local adaptation is a key process for the maintenance of genetic diversity and population diversification. A better understanding of the mechanisms that allow (or prevent) local adaptation constitutes a key in apprehending how and at what spatial scale it occurs. The production of resting stages is found in many taxa and reflects an adaptation to outlast adverse environmental conditions. Daphnia magna (Crustacea) can alternate between asexual and sexual reproduction, the latter being linked to dormancy, as resting stages can only be produced sexually. In this species, on a continental scale, resting‐stage production is locally adapted—that is, it is induced when the photoperiod indicates the imminence of habitat deterioration. Here, we aimed to explore whether selection is strong enough to maintain local adaptation at a scale of a few kilometers. We assessed life‐history traits of 64 D. magna clones originating from 11 populations of a metapopulation with permanent and intermittent pool habitats. We found large within‐ and between‐population variation for all dormancy‐related traits, but no evidence for the hypothesized higher resting‐stage production in animals from intermittent habitats. We discuss how gene flow, founder events, or other forms of selection might interfere with the process of local adaptation.     

Maternal effects after sexual reproduction in Daphnia magna

In this study we investigated the relationship between maternalsize and size of ephippial hatchlings in Daphnia magna. We observedthat larger females produce larger ephippia; these larger ephippiahave a higher hatching probability, yield larger neonates, whichin turn yield larger adults, producing more eggs. We interpretthese observations as an explanation why older and larger femalesof D.magna continue to produce ephippia despite a higher predationrisk.     

Variation in ephippial buoyancy in Daphnia pulicaria

1. Freshwater zooplankton often produce diapausing eggs to survive environmental stress. The diapausing eggs of Daphnia (Crustacea, Cladocera) are encased in an ephippium that either floats at the surface or sinks to the sediment. These two types of ephippia may represent different strategies between spatial (floaters) and temporal (sinkers) dispersal of offspring. 2. We observed floating and sinking characteristics of ephippia obtained from eight lakes. We then conducted an experiment with 26 Daphnia pulicaria clones obtained from six of these lakes and observed the production of buoyant and non‐buoyant ephippia under high and low food conditions. 3. Ephippia were more often non‐buoyant than buoyant both from females caught in nature and those reared in the laboratory. The experiment revealed that each clone was able to produce both types of ephippia, but that there was considerable among‐clone variation in the percentage of non‐buoyant ephippia produced. 4. We conclude that production of non‐buoyant versus buoyant ephippia may be driven by both genetic and environmental factors.     

QTL analysis of the photoperiodic response and clinal distribution of period alleles in Nasonia vitripennis

In seasonal environments, organisms synchronize their life cycle with the annual cycle of environmental factors. In many insect species, this includes a diapause response: a timed dormant stage that allows to survive harsh winter conditions. Previously, we have shown that larval diapause in the parasitic wasp Nasonia vitripennis is induced by the mother upon exposure to a threshold number of short photoperiods (named switch point) and diapause response follows a latitudinal cline in natural populations. Here, we present a QTL analysis using two lines derived from the extremes of this clinal distribution: a northern line from Oulu, Finland and a southern line from Corsica, France. A genomic region on chromosome 1 and one on chromosome 5 were found to be associated with photoperiodic diapause induction. Interestingly, these regions contain the putative clock genes period, cycle (chromosome 1) and cryptochrome (chromosome 5). An analysis of period polymorphisms in seven European populations showed a clinal distribution of two main haplotypes that correlate with the latitudinal cline for diapause induction.     

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

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

Diapause dynamics of two diaptomid copepod species in a large lake

Skistodiaptomus oregonensis and Leptodiaptomus minutus produce diapausing eggs in a large lake, Oneida Lake, in New York State. The timing of the switch from production of subitaneous (immediately hatching) eggs to diapausing eggs for both species is in October. This timing is consistent with a pattern, reviewed here, for other populations of diaptomid copepods: populations living in large lakes tend to begin production of diapausing eggs later in the season than those living in small lakes. Populations living in temporary ponds tend to switch still earlier in the season. All populations reviewed here live in the north temperate zone. The sediments of Oneida Lake contain densities of diaptomid diapausing eggs on the order of 10⁵ m^–2 per cm below the sediment surface down to 5 cm. Below this sediment depth, egg densities decline. The highest egg densities were found in sediments under the deepest water. Diapausing eggs of L. minutus survive in the sediments at least two years, as shown by the recovery of the population after a year in which no new diapausing eggs were produced, and probably for two or more decades. Long-term dormancy can have the effect of ensuring the continuation of a population through periods of poor recruitment, and can help create conditions for the coexistence of competing species. Other investigators have suggested that S. oregonensis and L. minutus are competitors in other lakes when they co-occur.     

Adaptive latitudinal cline of photoperiodic diapause induction in the parasitoid Nasonia vitripennis in Europe

Living in seasonally changing environments requires adaptation to seasonal cycles. Many insects use the change in day length as a reliable cue for upcoming winter and respond to shortened photoperiod through diapause. In this study, we report the clinal variation in photoperiodic diapause induction in populations of the parasitoid wasp Nasonia vitripennis collected along a latitudinal gradient in Europe. In this species, diapause occurs in the larval stage and is maternally induced. Adult Nasonia females were exposed to different photoperiodic cycles and lifetime production of diapausing offspring was scored. Females switched to the production of diapausing offspring after exposure to a threshold number of photoperiodic cycles. A latitudinal cline was found in the proportion of diapausing offspring, the switch point for diapause induction measured as the maternal age at which the female starts to produce diapausing larvae, and the critical photoperiod for diapause induction. Populations at northern latitudes show an earlier switch point, higher proportions of diapausing individuals and longer critical photoperiods. Since the photoperiodic response was measured under the same laboratory conditions, the observed differences between populations most likely reflect genetic differences in sensitivity to photoperiodic cues, resulting from local adaptation to environmental cycles. The observed variability in diapause response combined with the availability of genomic tools for N. vitripennis represent a good opportunity to further investigate the genetic basis of this adaptive trait.     

Population differences in the timing of diapause: adaptation in a spatially heterogeneous environment

Summary Populations of the planktonic copepod, Diaptomus sanguineus, live in permanent and temporary freshwater ponds in Rhode Island. All ponds in which they occur become uninhabitable at some time during the year, but the nature and timing of the harsh period varies both spatially and temporally. Females produce discrete clutches either of subitaneous eggs which hatch immediately or of diapausing eggs which hatch the following season. The two egg types show distinct chorion morphologies under transmission electron microscopy. In permanent ponds the copepods start making diapausing eggs in March, one month before rising water temperatures induce planktivorous sunfish to become active. In temporary ponds diapausing eggs are produced, in a complex pattern from May to July, before the water disappears in late summer or early fall.We investigated the spatial scale at which D. sanguineus is adapted to this complex environment. In a reciprocal transfer experiment between temporary and permanent bodies of water, female copepods placed in new ponds made subies of water, female copepods placed in new ponds made subitaneous and diapausing eggs in the same sequence as control females retained in their home ponds. The copepod populations enter diapause at times appropriate for the local habitat conditions they experience, but inappropriate for other, nearby ponds. Transplanted females were unable to sense a change in pond type or to adjust egg production accordingly. We conclude that D. sanguineus populations are adapted to the specific conditions of isolated ponds rather than to a broader geographical region containing several pond types.     

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.     

Predator-induced diapause in Daphnia magna may require two chemical cues

The production of diapausing eggs by Daphnia magna stimulated by fish exudates can be explained as an anti-predator defence ensuring genome protection in periods of high risk from fish predation. The combined effects on the induction of D. magna diapause of an “alarm” chemical originating from injured conspecific prey and fish kairomones were tested. The results of the experiment showed that the cues when present together promote both the production of ephippial eggs and male formation, indicating their role in the synchronization of the entire mode of Daphnia sexual reproduction. Ephippial eggs were only produced in the presence of both fish kairomone and conspecific alarm chemicals, while male offspring occurred in the treatments where both, one or none of the cues were present. However, production of males was the highest when both cues were provided. D. magna responded similarly to the tested cues whether or not the hypothetical alarm substance associated with predator odour came from Daphnia specimens actually eaten by fish or from crushed conspecific individuals. However, chemicals from crushed chironomid larvae combined with fish kairomones did not induce a similar response in D. magna. The relative advantage of utilization of alarm cues or predator kairomones in the induction of defence responses in prey organisms is discussed. Received: 8 June 1998 / Accepted: 11 January 1999     

A model evaluating the contribution of environmental factors to the production of resting eggs in the rotifer Brachionus plicatilis

The production of resting eggs by the rotifer Brachionus plicatilis was tested at four salinities (9, 18, 27 and 36\%) and six concentrations of the alga Chlorella stigmatophora (0.25, 0.5, 1.0, 2.0, 4.0 and 6.0 × 10⁶ cells ml^–1). The results indicated that resting eggs were produced only at two salinities (9\% and 18\%) and that their number was affected by the amount of food provided. A model consisting of two generalized linear sub-models was built to evaluate the contribution of each of the tested food concentrations at the two salinities. The sub-models were used to distinguish between two different components of resting egg production: one related to the presence or absence of resting egg production, and the other to the number of resting eggs produced, given that production had occurred. Besides indicating the best combination of salinity and food concentration for obtaining large numbers of resting eggs, they revealed the contribution of internal population factors that were not controlled in the course of the experiment. The model identified the positive contribution of the relative number of females to males, and the negative association between high rotifer densities and the production of resting eggs. The results of the present study help in defining the optimal conditions for mass production of resting eggs, which are of potential importance in aquaculture.Deceased, September 1991.     

Development of microsatellite markers for Diaphanosoma dubium (Crustacea,Cladocera) and application to seasonal population dynamics

Diaphanosoma, the “tropical Daphnia”, is common and ubiquitous in South China. Like other ctenopods, Diaphanosoma has a reproductive mode similar to Daphnia’s, but its resting eggs are rarely observed and lack an ephippium. With limited dispersal and reduced buffer effect from resting egg banks, Diaphanosoma is expected to have a population genetic structure different from that of temperate Daphnia. To facilitate genetic comparison, we developed microsatellite markers using next-generation sequencing for the most common species in tropical and subtropical East Asia, Diaphanosoma dubium. Thirty-one polymorphic microsatellite markers were obtained, and 29 of them were efficient for the congeneric species D. excisum, D. orghidani, D. mongolianum and D. chankensis. The markers allowed intra- and interspecific genetic analysis, including population structure, hybridization and introgression. We used 11 selected microsatellite markers to analyze spatial and temporal heterogeneity of genetic diversity in four (sub)tropical D. dubium populations from two large reservoirs and two temporary ponds. In contrast to temperate Cladocera, higher genetic diversity in summer rather than in spring suggested weak contribution from resting eggs in spring. Clustering of DAPC and STRUCTURE analyses indicated a clear-cut genetic structure in the four populations. Variation partitioning revealed that water storage and depth were key factors in genetic differentiation. Within large reservoirs, we detected backward (reversing time) gene flow from resting egg banks. We conclude that resting eggs have an effective contribution to the genetic diversity in large water bodies during growing seasons and that large water bodies can host higher genetic diversity in summer due to environmental heterogeneity and high carrying capacity. Spatial and temporal heterogeneity of genetic diversity detected by our microsatellite markers showed the newly developed markers can be applied for further study of populations of D. dubium and other species of Diaphanosoma at a contemporary scale.