Effects of winter conditions on Daphnia dynamics and genetic diversity in a dimictic temperate reservoir

1. Winter conditions shape plankton dynamics and community composition in temperate regions, but their effect on dynamics and genetic composition of cyclical parthenogens like Daphnia is largely unclear. 2. For 5 years, we studied the dynamics, hatching from resting eggs and genetic structure of a D. galeata × longispina hybrid complex in a dimictic, temperate reservoir. Our main hypothesis was that higher spring densities and an earlier population peak will be observed after warmer winters, with a lower genetic diversity because of a lower contribution of resting eggs to population growth. 3. The study period could clearly be categorised into cold‐winter years (n = 3) and warm‐winter years (n = 2). Daphnia densities at the end of spring overturn were ～10‐fold lower after cold winters than after warm ones, but no pattern emerged concerning the timing and the height of the population peak in early summer. 4. Hatching intensity from resting eggs was higher and contributed up to 8.5% to Daphnia abundance in a cold‐winter year compared to a negligible contribution in a warm‐winter year. Consistent with this finding, new multilocus genotypes (MLGs) adding to the overwintering stock after the end of spring overturn and presumably originating from resting eggs increased genetic diversity and attained high frequencies within the population only after a cold winter. New MLGs were recorded also after warm winters, but they never gained dominance and no shift in genetic diversity was observed. However, genetic diversity was not generally reduced after warm winters. 5. Our results confirm earlier findings that winter conditions have only a limited effect on the main growth phase and the peak of Daphnia during late spring and early summer. However, winter conditions determine the contribution of resting eggs to the population development, which may profoundly alter the genetic composition of the population compared to the previous season.     

Can overwintering versus diapausing strategy in <Emphasis Type="Italic">Daphnia</Emphasis> determine match–mismatch events in zooplankton–algae interactions?

Mismatches between predator and prey due to climate change have now been documented for a number of systems. Ultimately, a mismatch may have far-reaching consequences for ecosystem functioning as decoupling of trophic relationships results in trophic cascades. Here, we examine the potential for climate change induced mismatches between zooplankton and algae during spring succession, with a focus on Daphnia and its algal food. Whereas the development of an overwintering population of daphnids may parallel shifts in phytoplankton phenology due to climate warming, changes in the photoperiod–temperature interaction may cause the emerging population of daphnids to hatch too late and mismatch their phytoplankton prey. A decoupling of the trophic relationship between the keystone herbivore Daphnia and its algal prey can result in the absence of a spring clear water phase. We extended an existing minimal model of seasonal dynamics of Daphnia and algae and varied the way the Daphnia population is started in spring, i.e., from free swimming individuals or from hatching resting eggs. Our model results show that temperature affects the timing of peak abundance in Daphnia and algae, and subsequently the timing of the clear water phase. When a population is started from a small inoculum of hatching resting eggs, extreme climate warming (+6°C) results in a decoupling of trophic relationships and the clear water phase fails to occur. In the other scenarios, the trophic relationships between Daphnia and its algal food source remain intact. Analysis of 36 temperate lakes showed that shallow lakes have a higher potential for climate induced match–mismatches, as the probability of active overwintering daphnids decreases with lake depth. Future research should point out whether lake depth is a direct causal factor in determining the presence of active overwintering daphnids or merely indicative for underlying causal factors such as fish predation and macrophyte cover. Priority program of the German Research Foundation—contribution 5.     

Response of diapausing eggs hatching to changes in temperature and the presence of fish kairomones

It has been hypothesized that the production of diapausing eggs in Daphnia can be induced by fish kairomones. A population of Daphnia could survive severe predation using this predator avoidance strategy. However, in changing environments, diapausing eggs experience various temperature conditions, and hatchlings at emergence may be exposed to the same predation risks as their mothers. Therefore, staying in diapause or an immediate response upon hatching to available environmental information could be important for hatchling survival. For this study, we investigated the impact of water temperature (10, 15, 20, and 25°C) in the presence and absence of fish kairomones (Lepomis macrochirus) on the hatching success of resting eggs (D. galeata). Results show that no diapausing eggs hatched at the lowest temperature (10°C), and the highest hatch percentage occurred at 15°C. Although higher water temperatures reduced hatching success, diapausing eggs hatched more quickly. The number of hatchlings was significantly higher after exposure to fish kairomones, and this was more noticeable at higher temperatures (20 and 25°C). The present results suggest that the diapausing eggs were produced as a predator avoidance strategy in Daphnia; however, the presence of fish works as a positive signal to increase hatchlings when the diapausing stage is terminated.     

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.     

Are they still viable? Physical conditions and abundance of <Emphasis Type="Italic">Daphnia pulicaria</Emphasis> resting eggs in sediment cores from lakes in the Tatra Mountains

All species of Daphnia (Cladocera) produce, at some stage in their life cycle, diapausing eggs, which can remain viable for decades or centuries forming a “seed bank” in lake sediments. Because of their often good preservation in lake sediment, they are useful in paleolimnology and microevolutionary studies. The focus of this study was the analysis of cladoceran resting eggs stored in the sediment in order to examine the ephippial eggs bank of Daphnia pulicaria Forbes in six mountain lakes in the High Tatra Mountains, the Western Carpathians (northern Slovakia and southern Poland). Firstly, we analyzed distribution, abundance and physical condition of resting eggs in the sediment for their later used in historical reconstruction of Daphnia populations by genetic methods. To assess changes in the genetic composition of the population through time, we used two microsatellite markers. Although DNA from resting eggs preserved in the High Tatra Mountain lake sediments was extracted by various protocols modified for small amounts of ancient DNA, DNA from eggs was not of sufficient quality for microsatellite analyses. Distribution curves of resting eggs from sediment cores correspond to the environmental changes that have occurred in the High Tatra Mountains area during last two centuries (atmospheric acid deposition, fish introduction) and demonstrate their influence on natural populations. Evaluation of ephippia physical condition (the most common category was empty ephippial covers) suggests that the majority of resting eggs hatched to produce a new generation of Daphnia or may be due to failed deposition of resting eggs by Daphnia to the chitinous case. In conclusion, age, low quantity and poor physical condition of resting eggs from these Tatra lake sediments proved to be unsuitable not just for use in genetic analyses, but also the possibilities of autogenous restoration of Daphnia populations from the resting egg banks in the Tatra sediments are negligible.     

Influence of overwinteringDaphnia on spring zooplankton communities: An experimental study

To evaluate the influence of overwintering individuals of zooplankton on spring zooplankton communities, the dynamics of zooplankton communities with or without overwintering individuals were observed in experimental ponds from fall to spring. An insecticide, carbaryl, was used to regulate the overwintering individuals. In ponds which received insecticide applications in November or January, all cladoceran and rotiferan species were eliminated by the treatments and did not reappear until late March or early April, even when the chemical disappeared rapidly. The low water temperature may delayed the establishment of the populations from resting eggs. In these ponds, populations of various cladoceran and rotiferan species, which seemed to be originated from resting eggs, were built up in the spring. In control ponds,Daphnia ambigua orD. longispina overwintered as juveniles and adults and established a large spring population earlier than other cladocerans and rotifers overwintering as resting eggs. The latter zooplankters did not increase in the spring probably because their growth was suppressed by the precedingDaphnia species through competition. In nature, even if the number of overwintering individuals is small, they may have a potential to build up a large population earlier than the individuals hatching from resting eggs. As a result, the species which have overwintered as individuals seem to predominate in the spring and have a large influence on the spring zooplankton community.     

Fish as vectors in the dispersal of Bythotrephes cederstroemi: diapausing eggs survive passage through the gut

• 1 Bythotrephes cederstroemi (Crustacea: Onychopoda: Cercopagidae) is an introduced invertebrate predator currently spreading through the Laurentian Great Lakes region of North America. We examined a previously unsuspected way in which B. cederstroemi may be dispersed by fish by their consumption of diapausing eggs.
• 2 Ninety‐four percentage of the mature B. cederstroemi diapausing eggs consumed by fish were egested apparently intact. This proportion is considerably above previous estimates for the ephippial eggs of Daphnia. The hatching success of diapausing eggs was compared among four categories: (a) eggs released naturally by B. cederstroemi (control, 73% hatched) (b) eggs released during ‘stressful confinement’ (46% hatched) (c) eggs dissected from dead females (13% hatched) and (d) eggs recovered from faecal pellets following consumption by fish (viable gut passage experiment, 41% hatched).
• 3 Samples of small fish and B. cederstroemi were collected simultaneously. Examination of gut contents revealed that fish contained B. cederstroemi diapausing eggs and that B. cederstroemi bearing resting eggs were consumed selectively over those without eggs. Moreover, fish selected B. cederstroemi bearing mature rather than immature diapausing eggs.
• 4 The fact that diapausing eggs survive gut passage is important for the dispersal of B. cederstroemi. Fish often move between the pelagic and littoral zones of lakes and may thus disperse diapausing eggs widely. Fish may also move between lakes connected by river systems and can be caught and passively dispersed by anglers or piscivorous birds. Our results demonstrate the potential for fish to act as vectors in the spread of B. cederstroemi.

     

DNA extraction and amplification methods for ephippial cases of Daphnia resting eggs in lake sediments: a novel approach for reconstructing zooplankton population structure from the past

This study describes a method of DNA extraction and amplification for ephippial cases of Daphnia resting eggs from lake sediment. Recent studies have reconstructed succession records of Daphnia species by genetically analyzing Daphnia resting eggs stored in lake sediments and revealed changes in dominant Daphnia species that correspond well with environmental changes. However, this approach is not applicable to lakes where most of the resting eggs in the sediment have already hatched out. We modified conventional methods for DNA extraction and amplification to enable genetic analyses of the ephippial case that envelops and protects the resting eggs, and we compared the performance of the modified method to the conventional one. We confirmed that we could efficiently analyze the sequences of the ephippial cases collected in a sediment core using our modified method. It enables us to reconstruct changes in genetic structure of Daphnia populations regardless of hatching rates of the resting eggs.     

Reproduction strategies of <Emphasis Type="Italic">Daphnia pulicaria</Emphasis> population in a high mountain lake of Southern Spain

For three consecutive years, a population study of Daphnia pulicaria was undertaken in Río Seco Lake, a small high-mountain lake, in order to elucidate the reproductive strategies adopted by Daphnia in this system. Daphnia appears to colonize this lake every spring by hatching from ephippia and reproduce by means of subitaneous (non-diapausing) and ephippial (diapausing) eggs. D. pulicaria in this lake is an obligate parthenogenetic population. There is a short time period for subitaneous egg production and a much longer period for ephippial egg production. The contribution of subitaneous eggs to Daphnia population density and structure appears to be low. Diapause onset showed a high temporal synchronization in the three studied years in Río Seco Lake, and day-length emerged as the main cue triggering diapause onset and the main explanatory factor for the proportion of ephippial females observed. The development and reproduction of D. pulicaria in Río Seco Lake involves taking a gamble on resting forms to guarantee inter-annual Daphnia persistence in the lake, giving priority to investment in future generations. Guest editor: Piet Spaak Cladocera: Proceedings of the 7th International Symposium on Cladocera     

Local variation in overwintering stages ofPanonychus akitanus Ehara (Acarina: Tetranychidae)

The overwintering behaviour ofPanonychus akitanus Ehara was compared in a population (Sapporo) overwintering as the egg and the adult female and in a population (Tomakomai) overwintering in the egg stage only. In both populations, winter eggs were larger in size and darker in colour than summer eggs, and females laying winter eggs showed clear differences in body colour and behaviour from summer females. Both populations produced winter eggs in response to short photoperiods, and the critical photoperiod was between 13.0 and 13.5 h in the Sapporo population and between 13.5 and 14.0 h in the Tomakomai population. The hatchability of winter eggs of the Sapporo population collected from the field began to increase in November, and that of the Tomakomai population in December. In the Sapporo population, a 40-day or more exposure to 5°C promoted hatching of winter eggs, and a 50-day exposure of females which had previously laid some winter eggs to 5°C induced the production of summer eggs. Females of the Sapporo population laid winter eggs in autumn, and then themselves overwintered as well, and produced summer eggs in the following spring. These females were called winter-habit females.The winter eggs of both populations were diapausing. However, the winter-habit females of the Sapporo population were not reproductively inactive and their condition of diapause was not certain. The ecological significance of the winter-habit females in the Sapporo population is discussed.     

Patterns in the abundance,phenology, and hatching of the resting egg stage of the invasive zooplankter <Emphasis Type="Italic">Bythotrephes longimanus</Emphasis>: implications for establishment

To examine how dormancy contributes to the establishment and persistence of Bythotrephes longimanus, we investigated resting egg production and hatching in relation to the demography of the planktonic stage and environmental conditions in Island Lake Reservoir (USA). During a 3-year study, the largest contribution to the egg bank occurred in autumn and most eggs hatched in spring, but we also detected some resting egg production and hatching in summer. The difference between summer and late autumn densities of eggs in sediments averaged 47% (range 0–98%) for 18 sites throughout the reservoir, which was similar to experimental estimates of in situ hatching fraction of 67% for eggs in the spring and summer following their production. Based on emergence traps, neonates hatch in the field during May and June. We estimated mortality rates of 64% for resting eggs and embryos, and 59% for newly emerged neonates. Although hatching fraction saturated at the same level, eggs incubated offshore hatched later than those nearshore where water temperature was warmer and light was detectable at the sediment surface. Low dissolved oxygen concentration did not significantly reduce hatching fraction but resulted in some eggs that initiated development but failed to hatch. Collectively, our results demonstrate substantial annual turnover in the resting egg bank of B. longimanus and high mortality of resting eggs during recruitment from the egg to the first molt of the planktonic stage. These patterns suggest that propagule pressure in the form of resting eggs requires large numbers for establishment, and that considerable post-establishment resting egg production is necessary for inter-annual persistence.     

Hatching and viability of rotifer diapausing eggs collected from pond sediments

1. Planktonic rotifers inhabiting variable environments produce diapausing eggs that accumulate in the sediment of lakes and ponds, forming egg banks that may withstand adverse periods. A common assumption in zooplankton diapausing egg bank studies is to count as viable all eggs in the sediment that look healthy. This assumption should be challenged by asking how effectively ‘healthy‐looking’ eggs represent viable eggs. 2. In this study, viability of more than 1100 ‘healthy‐looking’ diapausing eggs belonging to the Brachionus plicatilis species complex was assessed in a laboratory hatching experiment. Eggs were collected at different depths from sediment cores obtained from 15 ponds located in coastal and inland areas of Eastern Spain. 3. Only approximately one half of the ‘healthy‐looking’ diapausing eggs hatched after incubation in experimental conditions. Almost all the hatchlings (99.4%) survived to maturity. The proportion of ‘healthy‐looking’ diapausing eggs that hatched varied among areas and among ponds within area, and substantially declined with sediment depth. Most of the hatchlings (88%) were obtained from the uppermost 2 cm of sediment. ‘Healthy‐looking’ eggs from upper sediment layers hatched after significantly shorter incubation times than eggs recovered from deeper layers. 4. Both decreased hatching success and increased incubation time for hatching with sediment depth suggest that older ‘healthy‐looking’ eggs are less responsive to hatching stimuli and could become unviable. However, the strong correlation found between the number of ‘healthy‐looking’ eggs and the number of hatchlings indicates that the abundance of ‘healthy‐looking’ eggs is a good index of egg bank viability.     

Growth and fecundity of Daphnia after diapause and their impact on the development of a population

Laboratory and field investigations revealed that the life history traits of exephippial and parthenogenetic generations of Daphnia differ substantially. Daphniids hatching from resting eggs grow faster and their definitive body sizes are bigger than of hatchlings from subitaneous eggs. Size at maturity for exephippial animals is significantly larger. In spite of this, they mature a few days earlier than parthenogenetic females. In this study, the difference was 3–4 days for the laboratory experiments and 1–3 days for the field. Fecundity of the exephippial generation is markedly higher. Here, the clutch size for this generation was up to 3.5–4.0 times as large as for the parthenogenetic generation. Moreover, obtained results suggest that the relationship between clutch size and body length for both generations differ significantly.Estimates of the intrinsic rate of increase for field Daphnia populations demonstrated that life history traits of exephippial animals lead to a two or threefold higher rate of increase in the conditions of invertebrate predation pressure. Under moderate fish pressure, obtained r values for the daphniids hatching from resting eggs were larger than those from subitaneous. High growth rate of exephippial females is disadvantageous only under the conditions of severe pressure by fish. Obtained results suggest that hatchlings from diapausing eggs an acceleration of population increase by several times during the beginning of the development of a population with periodical re-establishment from resting eggs.     

Implications of resting eggs of zooplankton for the paradox of enrichment

In this study, we numerically investigated to what extent introducing resting-egg dynamics would stabilize simple Daphnia–algae consumer–resource models. In the models, the density of viable resting eggs was explicitly expressed, and we assumed that zooplankton produced resting eggs seasonally or in response to food deficiency and that resting eggs hatched seasonally. The models predicted that, although the paradox of enrichment was not completely resolved (i.e., the system was destabilized by eutrophication), we found the following conditions under which the stabilizing effects of resting eggs would be significantly large: (1) resting eggs are produced seasonally (rather than in response to food deficiency), (2) the annual average allocation ratio to resting eggs is large, and (3) the annual average hatching rate of resting eggs is low. The results suggest that resting-egg dynamics can significantly reduce the paradox of enrichment within the biologically meaningful parameter space and contribute to the stability of plankton community dynamics.     

Role of parthenogenetic natality and emergence from diapausing eggs in the dynamics of some rotifer populations

There are few quantitative data on the role of emergence from diapausing eggs in population dynamics of natural populations of zooplankton species; to our knowledge, all these concern copepods and cladocerans. We present here direct estimates of emergence from bottom resting eggs for another important category of freshwater zooplankton, namely rotifers. Three populations of rotifers of the genus Brachionus were studied in a lake. During the study period 10 population increases, each corresponding to an individual sampling interval, were detected. For each interval, emergence from immediately hatching, parthenogenetic eggs calculated on the basis of the Edmondson-Paloheimo model and emergence from diapausing bottom eggs determined in short-term experiments were estimated and compared to each other. We found that three of the population increases observed are entirely explained by parthenogenetic natality. In contrast, emergence from diapausing eggs can, on its own, account for none of population increases. For two population increases, however, it accounts for that part of population growth which remains unexplained by the parthenogenetic natality. For rotifer populations studied, emergence from diapausing eg eggs is generally less important than parthenogenetic births, when both are regarded as an immediate cause of population growth. This is in sharp contrast to the data available for some crustaceans (De Stasio, 1990) where the role of emergence from diapausing eggs in population dynamics has been clearly shown.     

A major shift in Daphnia genetic structure after the first ice‐free winter in a German reservoir

1. Climate warming may cause disruption of trophic linkages in aquatic ecosystems and lead to changes in abundance and genetic structure of zooplankton populations. We monitored the community of the Daphnia galeata‐hyalina hybrid complex in the Saidenbach Reservoir (Saxony, Germany) using allozyme electrophoresis for three consecutive years (2005–07), including one (2007) following an unusually warm winter that prevented the formation of ice cover for the first time in the history of the reservoir. 2. Genetic composition during the 2007 season differed substantially from the two preceding years that experienced the usual 3‐month ice period. Three abundance peaks in June, July and October 2007 were dominated by hybrids of Daphnia galeata x hyalina, whereas in the 2005 and 2006 seasons two peaks in June and September were dominated by Daphnia hyalina genotypes. 3. The genetic composition of the pool of diapausing eggs produced in autumn and the rate of change of genotype abundance during the following spring indicate recruitment of the D. hyalina subpopulation from ex‐ephippial animals during the spring population increase. 4. The differing potential to contribute to the overwintering animal pool or to the inoculum from diapausing eggs was confirmed by results from laboratory life‐table experiments. Daphnia galeata clones survived longer and produced parthenogenetic offspring under winter conditions, whereas D. hyalina clones showed a shorter lifespan and produced resting eggs. 5. Our results indicate a profound role of recruitment strategy in the observed shift in genetic composition. Increasing winter temperatures predicted in the context of climate change may thus favour overwintering animals, leading to an increase in the contribution of these genotypes to the population. Such microevolutionary processes may dampen possible seasonal mismatches between daphnid populations and their food or predator populations.     

Genetic variability and transgenerational regulation of investment in sex in the monogonont rotifer Brachionus plicatilis

In cyclical parthenogens such as aphids, cladocerans and rotifers, the coupling between sexual reproduction and the production of resting stages (diapausing eggs) imposes strong constraints on the timing of sex. Whereas induction of sex is generally triggered by environmental cues, the response to such cues may vary across individuals according to genetic and nongenetic factors. In this study, we explored genetic and epigenetic causes of variation for the propensity for sex using a collection of strains from a Spanish population of monogonont rotifers (Brachionus plicatilis) in which variation for the threshold population density at which sex is induced (mixis threshold) had been documented previously. Our results show significant variation for the mixis threshold among 20 clones maintained under controlled conditions for 15 asexual generations. The effect of the number of clonal generations since hatching of the diapausing egg on the mixis ratio (proportion of sexual offspring produced) was tested on 4 clones with contrasted mixis thresholds. The results show a negative correlation between the mixis threshold and mixis ratio, as well as a significant effect of the number of clonal generations since fertilization, sex being repressed during the first few generations after hatching of the diapausing egg.     

Single dietary amino acids control resting egg production and affect population growth of a key freshwater herbivore

The enormous success of the genus Daphnia in freshwater ecosystems is at least partially due to their cyclical parthenogenetic life cycle, in which asexual and sexual reproduction alternate periodically. This temporal change between reproductive strategies allows for (1) rapid population growth via subitaneously developing eggs when environmental conditions are appropriate and (2) the maintenance of genetic diversity via sexual reproduction and the production of resting eggs when environmental conditions deteriorate. We show here that dietary amino acids are involved in triggering the switch between reproductive modes in Daphnia pulex. Supplementation experiments demonstrate that specific dietary amino acids, in particular arginine and histidine, avert crowding-induced resting egg production, enhance subitaneous reproduction by increasing algal food quality and, as a combined effect of both processes, increase population growth rates. These findings suggest that the availability of single dietary amino acids potentially affects the seasonal dynamics and long-term persistence of Daphnia populations in the field, which may have consequences for the efficiency of carbon transfer and thus the trophic structure of freshwater food webs.     

Deterioration patterns in diapausing egg banks of Brachionus (Müller, 1786) rotifer species

Rotifers are cyclical parthenogens that produce sexual diapausing eggs at some stage in their life cycle. These eggs are encysted embryos that remain viable for extended periods in lake and pond sediments, thus acting as an egg bank with many ecological and evolutionary consequences. Despite its importance to rotifer evolution, there are no studies on resting egg deterioration and associated processes in natural environments. In this study, more than 4000 diapausing eggs of species from the Brachionus plicatilis complex, which includes several closely related cryptic species, were collected from different sediment depths in 15 ponds in eastern Spain and were classified according to three features thought to be related to their viability: shell integrity, embryo size, and embryo colour. A positive association was found between embryo size and hatching success in those eggs having an intact shell. Diapausing eggs that showed good shell integrity and no more than a 25% reduction of multinuclear embryo maximum size were classified as healthy and 98.9% hatched. Darkening of diapausing egg embryo was an indicator of viability loss. A decreasing frequency of healthy-looking diapausing eggs was observed with increasing sediment depth, although some exceptions were found.