Low crowding threshold for induction of sexual reproduction and diapause in a Patagonian rotifer

1. This study investigates the basis and ecological significance of the extremely high propensity for mixis (sexual reproduction) observed in laboratory populations of Brachionus calyciflorus from a temporary pond in Patagonia. 2. Experiments with stem females hatched from resting eggs showed that these females were exclusively amictic but produced mictic daughters even at very low population densities. In six experiments, newly hatched stem females were cultured individually in different volumes (1.5, 12, 40 and 150 mL). The percentage of mictic daughters (mixis ratio) was high in the smaller volumes (e.g. 44–83% in 1.5 mL) and lower in the largest volume (6–21% in 150 mL). A regression analysis combining the data from these experiments showed a highly significant decrease in mixis ratio with volume and indicated that the lowest population density at which mixis still occurs (mixis threshold) would be 3.4 females L^?1 (95% CL 2.9–4.0 females L^?1). This value is considerably lower than mixis thresholds for other rotifers (25–250 females L^?1 for many species and 9000–477 000 females L^?1 for some Spanish Brachionus plicatilis). 3. In three additional experiments, stem females and their amictic daughters were cultured individually in 150 mL. The percentage of mictic daughters produced by these two generations of females was not significantly different, showing that the mixis response to crowding is not inhibited in the stem‐female generation. 4. Laboratory experiments showed that two common predators of the Patagonian B. calyciflorus (the calanoid copepod Parabroteas sarsi and the backswimmer Notonecta vereertbruggheni) each ate 60–70 B. calyciflorus predator^?1 per day and cleared all rotifers from c. 250 mL per day. Thus, a very low mixis threshold and high maximal mixis ratio may ensure production of some resting eggs soon after colonisation of the pond and before complete removal from the plankton. 5. Two laboratory experiments showed that resting eggs of the Patagonian B. calyciflorus hatched at variable rates (28 and 81%) after a brief diapause when kept in the conditions under which they were produced and oviposited (20–21 °C; L: D 16: 8). Early hatching of resting eggs from pond sediment may allow multiple periods of colonisation and resting egg production in a season. This may offset the fitness cost of limited population growth through female parthenogenesis in the face of unpredictable and abrupt risk of extinction because of predators.     

Effect of incubation and preservation on resting egg hatching and mixis in the derived clones of the rotifer Brachionus plicatilis

The marine rotifer Brachionus plicatilis typicus (Clone 8105A, Univ. of Tokyo) was cultured in 500 ml beakers to form resting eggs. Tetraselmis tetrathele was used as a culture food. Just after formation, resting eggs were exposed to various temperature (5–25 °C) and light regimes (24L: OD and OL : 24D). When eggs were exposed to light just after formation, the eggs hatched sporadically over a month. No hatching was observed for six months when eggs were preserved under dark conditions regardless of the temperature. These eggs hatched simultaneously after being exposed to light and eggs preserved at 5 °C showed twice as high hatching rate (40%) as that of eggs preserved at 15–25 °C (24%). Clones from resting eggs that were kept under different temperature and light regimes were reared individually to the third generation. Incubation at 25 °C with lighting produced the highest (5.4% and 5.2 %) rate of mictic females during their 2nd and 3rd generations, respectively. The lowest rates (0 and 1.5%) were found when the eggs were kept at 5 °C in total darkness for six months. A lower rate of amictic female production was found in clones with higher rates of mixis.     

Temperature conditions enhancing resting egg production of the euryhaline rotifer Brachionus plicatilis O. F. Müller (Kamiura strain)

We examined the effect of low temperature treatment(12°C), followed by transfer to highertemperature (25°C), on resting egg formation ofthe rotifer Brachionus plicatilis, Kamiurastrain. This strain has been mass cultured as livefeed at Kamiura Station (Japan Sea FarmingAssociation) for 9 years at 20°C without theappearance of sexual reproductive stages.After preculture in 20 l of 27 seawater at 12°C for 0, 10, 20, and 30 days,rotifers were inoculated into 0.5 l mass cultures andcultured at 25°C for 7–9 days. The inoculationdensities were changed from 20 to 400 ind. ml^–1,depending on mixis rate. Condensed and frozen Nannochloropsis oculata was fed to rotifers at thefeeding rate of 0.14 µg (dry weight)rotifer^–1day^–1. The control was cultured at12°C for the entire 36 day experiment. No mixisappeared and no resting eggs were produced when thelow temperature treatment was 0 or 10 days. However,mixis rates reached 50-60% after 20 or 30 days ofexposure to 12°C. The number of resting eggsproduced in these treatments reached 25,500 about 13 times higher than the control. Our resultssuggest that low temperature stimulated mictic femaleproduction and the transfer to the high temperatureaccelerated resting egg formation. This method may beuseful for producing resting eggs of rotifer strainsthat lack sexual reproduction in the common culturecondition at larval rearing facilities.     

Effect of diazinon on life stages and resting egg hatchability of rotifer Brachionus plicatilis

The effects of organophosphate pesticide, diazinon, on life history parameters and hatchability of resting eggs of rotifer Brachionus plicalitis were assessed. Newly hatched (<1 h-old) neonates were individually cultured in six varying concentrations (0/control, 0.1, 1.0, 2.5, 5.0 and 10.0 mg/l) of diazinon. The life history parameters such as time (h) the rotifers bear first egg and release first neonate, reproductive period, net reproductive rate, mixis, intrinsic rate of population increase, and life span were evaluated. Results showed that among the life history parameters, the time the rotifers took to release neonates is the most sensitive, giving the lowest EC₅₀ value of 1.24 mg/l. The fecundity of maternal females, amictic and mictic daughters was also investigated. Rotifers exposed to 10.0 mg/l produced significantly fewer amictic daughters, and at this concentration, rotifers did not produce any mictic daughter. At 5.0 mg/l, the number of male offspring was significantly lower than the control. Furthermore, the hatchability of resting eggs produced by the rotifers was evaluated when exposed to diazinon: from birth until they produced resting eggs (early development); during late developmental stage of resting eggs (before diapause); and during diapausing stage. The hatchability of the resting eggs was not affected when exposure was timed at late developmental and diapausing stages. Overall results showed that even though amictic females reproduced normally in the presence of low-concentration of diazinon, sexual reproduction is severely affected, especially the hatchability of resting eggs when the exposure was timed on its early developmental stages. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont & R. Rico-Martínez Advances in Rotifer Research     

Effect of food concentration on the production and viability of resting eggs of the rotifer Brachionus: implications for the timing of sexual reproduction

1. Life‐table experiments with Brachionus calyciflorus test several hypotheses related to the idea that sexual reproduction in monogonont rotifers should occur when food resources are favourable. 2. The food concentration necessary for a fertilised mictic female to produce one phenotypically normal resting egg was higher than that for an amictic female to produce one daughter. At the lowest concentration of Cryptomonas erosa (1.25 × 10³ cells mL^?1), the lifetime fecundity of these two types of females was 0.9 and 1.4, respectively. 3. The lifetime fecundity of both fertilised mictic females and amictic females increased with food concentration to 3.4 resting eggs and 15.2 daughters female^?1, respectively. The approach to maximal fecundity with increasing food concentration was more rapid for fertilised mictic females, such that their lifetime fecundity relative to that of amictic females gradually decreased from 0.64 (at 1.25 × 10³ C. erosa mL^?1) to 0.22 (at 2.5 × 10⁴ C. erosa mL^?1). 4. The probability of a fertilised mictic female producing one or more abnormal resting eggs during her lifetime was high (approximately 75%). The mean proportion of abnormal eggs produced per female varied among the different food‐concentration treatments (26–38%) but was not higher at the low food concentrations. 5. The proportion of normal resting eggs that hatched was high (51–71%); those produced at low food concentrations were no less likely to hatch than those produced at high food concentrations. No abnormal resting eggs hatched. 6. The probability of a fertilised mictic female producing an abnormal resting egg increased rapidly with her age at all food concentrations. The probability of a normal resting egg hatching declined with maternal age at the low food concentration in one of two experiments. 7. The results support the idea that induction of mictic females should occur when food resources are good. Coincidence of sexual reproduction with low food availability risks low production of resting eggs for several reasons. Population size may be small, with a low probability of encounters between young mictic females and males, and fertilised mictic females may be unable to mature and produce resting eggs.     

Delayed mixis in rotifers: an adaptive response to the effects of density-dependent sex on population growth

In most cyclically parthenogenetic life cycles, sex is neededto produce resting stages. In several species of cyclicallyparthenogenetic rotifers, some generations of clones are notresponsive to a density-dependent signal that triggers sexualfemale production. These unresponsive rotifers hatch from restingeggs and typically pass 8–12 generations of female parthenogenesisbefore becoming receptive to the mixis signal. We addressedthe selection for mixis delay using a simulation model. A delayof sexual reproduction could increase population growth throughparthenogenesis and thus the number of resting eggs ultimatelyproduced. In a monomorphic population without mixis delay, wedetermined the optimal ratio of mictic to amictic females (mixisratio) to be 45%, and the optimal population density thresholdfor induction of mictic females (mixis threshold) to be 82 rotifersL^–1. This mixis pattern, however, was not an evolutionarilystable strategy. A mixis ratio of 14% and threshold of 70 rotifersL^–1 proved to be resistant to invasion by other mixispatterns. When we gave this phenotype a mixis delay of 8–10days, it could invade a population with the same mixis pattern,but lacking a mixis delay. The advantage of delaying mixis wasrelatively small, suggesting that a polymorphism is possible.     

Sexual reproduction and diapause of Hexarthra sp. (Rotifera) in short-lived ponds in the Chihuahuan Desert

1. In the life cycle of monogonont rotifers it is generally assumed that diapausing eggs invariably hatch into amictic stem females which produce female offspring parthenogenetically. Diapausing eggs are only produced by later generations after sexual reproduction has been induced by environmental cues. 2. We show that populations of an undescribed Hexarthra species inhabiting small temporary ponds in the Chihuahuan Desert deviate from this life cycle pattern. These ponds may dry within days and up to 85% of females were mictic. Females producing male offspring and diapausing eggs were observed 1 or 2 days, respectively, after ponds had filled with water. 3. Under laboratory conditions, 7–46% of females hatching from re‐hydrated sediments were sexual. Male offspring of these females can fertilise other mictic stem females leading to diapausing egg formation. In laboratory experiments, females produced fully developed diapausing eggs within 1.9 days at 20 °C and 1.2 days at 30 °C. 4. In addition, embryonic development time (1.1–0.3 days at temperatures between 12 and 30 °C) and juvenile period (2.1–0.5 days for the same temperature range) are shorter than those of other rotifer species. In short‐lived habitats, the potential for rapid population development and production of new diapausing eggs may be crucial in the long‐term survival of populations.     

Virus morphological diversity and relationship to bacteria and chlorophyll across a freshwater trophic gradient in the Lake Michigan watershed

The effect of starvation on sexual reproduction in cyclic parthenogenetic rotifers has been studied using life history experiment. Short-time starvation of rotifers that experienced starvation immediately after hatching from resting eggs can cause high induction of sexual reproduction up to the 10th generation. However, it is not clear whether the induction of sexual reproduction can occur beyond the 10th generation. To investigate this phenomenon, we conducted a sex induction study using the monogonont rotifer Brachionus manjavacas. Newborn stem females were starved for 12 h, while controls were supplied with 7.0 × 10⁶ cells ml^?1 of Nannochloropsis oculata. In a life history experiment, the rotifers were individually cultured in 96-well microplates containing 0.2 ml of seawater (22 ppt) in each well at 25 °C with daily feeding thereafter. Mixis induction in offspring from starved stem females was significantly higher than in those from non-starved stem females up to the 40th generation. The effect of accumulative generations increased mixis induction up to the 20th generation. Effect on future generations of the rise in mixis ratio by the starvation to stem females may facilitate colonization by favoring population growth via female parthenogenesis and by decreasing food requirements for survival and reproduction.     

Influence of the age of algae fed to rotifers (Brachionus plicatilis O.F. Müller) on the expression of mixis in their progenies

Summary The sequence of the appearance of mixis in the rotifer Brachionus plicatilis was followed among the descendents of amictic rotifers transferred from a high salinity media (40 S) to a low one (9 S). All the neonates that hatched from the amictic eggs, after being transferred to a low salinity, were amictic. Each one of these neonates was cultured individually and its offspring removed periodically every 8–10 h. It was observed that throughout their reproductive phase, these parental females retained their potential to produce either mictic or amictic offspring. All the first produced neonates developed into amictic females, but among those produced later, three patterns were prevalent. The prevalent pattern (type A) was one in which the probability of a neonate being mictic increased towards the middle of the parents' reproductive phase and was followed by a slow decline. In the second pattern (type B), the probability of a daughter being mictic was constant throughout the parents' reproductive phase. It is suspected that the quality of food supplied to the rotifers determines the appearance of patterns, A, B or C. It is postulated that the innate capacity of rotifers to undergo mixis is genetically controlled, while its expression is modulated by environmental conditions.     

Optimal rates of bisexual reproduction in cyclical parthenogens with density-dependent growth

This work explores theoretical patterns of reproduction that maximize the production of resting eggs and the long-term fitness of genotypes in cyclical parthenogens. Our focus is on density-dependent reproduction as it influences the consequences of a trade-off between producing amictic daughters – which reproduce parthenogenetically and subitaneously – and producing mictic daughters – which undergo meiosis and bisexual reproduction. Amictic females increase competitive ability and allow the population to achieve a larger size; mictic females directly contribute to population survival through harsh periods by producing resting eggs. Although morphologically indistinguishable, the two types of females differ greatly in their ecological and reproductive roles. What factors underlie the differential allocation of resources to produce amictic and mictic females? Using a demographic model based on readily accessible parameters we demonstrate the existence of a frequency of mictic females that will maximize the population's long-term fitness. This frequency, termed the optimal mictic ratio, m_o, is 1 ? (q/b)^1/2, where q is the mortality rate and b is the maximum birth rate. Using computer simulation we compared the fitness of a population with this constant mictic ratio with populations having multiple switches from complete parthenogenetic growth to complete allocation in mixis (mictic ratio either 0 or 1). Two important conclusions for optimal mixis in density-dependent growth conditions are: (1) intermediate mictic ratios are optimal, and (2) optimal mictic ratios are higher when habitat conditions are better. Physiological cues responding to differences in birth and death rates are common so that it is possible that populations may adjust their relative rates of mictic and amictic female production in response to environmentally induced changes to the optimum mictic ratio. Our analysis demonstrates that different patterns of mixis are expected in different type of habitats. Since the optimal mictic ratio is sensitive to the effects of a variety of environmental challenges, our model makes possible a new means to evaluate life history evolution in cyclical parthenogens.     

Variability for mixis initiation in Brachionus plicatilis

Deductions from both evolutionary models and inductive argumentation from empirical data support the notion of intraspecific variability for the initiation of sexual reproduction (mixis) within rotifer populations. In this study, we focus on the time and density at which mixis is initiated in a growing population. Cyclical parthenogenetic clones of Brachionus plicatilis established by hatching of resting eggs, isolated from a natural habitat, have been tested at the start of their sexual phase. Clones exhibited great variation for this trait, their time of switching to sexual reproduction being correlated with population density. Most of the variation for mixis initiation has either low or no heritability and is caused by individual environmental factors.     

Maternal Effect by Stem Females in <Emphasis Type="Italic">Brachionus plicatilis</Emphasis>: Effect of Starvation on Mixis Induction in Offspring

We examined whether starvation during the initial period of life in stem females affected reproductive characteristics of the offspring. Starvation treatment had different effects on rotifers hatched from resting eggs and those hatched from amictic eggs. When stem females experienced starvation after hatching, this induced a higher percentage of mixis in their offspring. When the same starvation treatment was applied to rotifers hatched from amictic eggs, there was no effect on the induction of mixis. It is probable that stem females hatched from resting eggs have specific features that are vulnerable to unfavorable environmental conditions, and that these features can be inherited by their offspring through the maternal cytoplasm.     

Analysis of proteins in conditioned medium that trigger monogonont rotifer mictic reproduction

The initiation, rate, duration, and termination of reproduction in the rotifer life cycle are tightly regulated, but the molecular mechanisms are poorly understood. Environmental signals are sensed and transduced to chemical signals within rotifers to integrate asexual and sexual reproduction. Crowding, diet, and photoperiod are environmental signals used by various rotifer species to initiate sexual reproduction, but most advances in the last 10 years have been in discovering how rotifer crowding is a case of quorum sensing. Female brachionids release a protein into their medium that acts as a pheromone and accumulates to trigger mictic reproduction. Candidates for this mixis-inducing protein (MIP) have been isolated from rotifer-conditioned medium and characterized biochemically. A N-terminal partial amino acid sequence of a particularly promising 39 kD protein was used to prepare a polyclonal antibody. This antibody immunoprecipitated a 22 kD protein from rotifer-conditioned medium and inhibited mixis induction. Future advances will benefit from the production of transgenic rotifers to confirm the mixis-inducing action of this putative MIP gene.     

新安江水域(屯溪段)轮虫的种群增长研究

应用群体培养法对采自新安江水域(屯溪段)的萼花臂尾轮虫在4种不同温度(15℃、20℃、25℃和30℃)下的种群动态进行研究。结果表明,温度对轮虫的种群增长率和休眠卵产量均有显著影响(P<0.05)。随着培养温度的升高,轮虫的种群增长率逐渐增大;休眠卵产量在15℃时最小,其余温度下无显著差异。轮虫的总雌体密度在25℃最大,15℃最小;4温度下,雄体和产雄卵的混交雌体的生产量15℃下最小,25℃下最大;而产休眠卵的混交雌体的生产量在15℃下最小,20℃下最大。温度对轮虫的平均混交雌体百分率和平均混交雌体受精率均无显著影响。     

Effects of Juvenile Hormone and Precocene on the Reproduction of Brachionus calyciflorus

We studied the effects of juvenile hormone and precocene on reproduction of the rotifer Brachionus calyciflorus. Amictic females of B. calyciflorus that were 2‐4 hours old were exposed to different concentrations of juvenile hormone (0.004, 0.02, 0.1, 0.5, 2.5, 12.5 mg/L) and/or precocene (0.05, 0.25, 0.75, 3.75, 7.5 mg/L) for 24 h. They were then transferred to a new medium without hormone and checked every 2 h during the next 48 h, and thereafter monitored daily until the individual died. Precocene had no effects on the length of the rotifer juvenile period, hatching time of the first neonate, lifetime reproduction, or the mixis ratio. In contrast, juvenile hormone at 0.5, 2.5, and 12.5 mg/L significantly prolonged the juvenile period by 6.1, 9.2, and 8.6%, respectively. When 26‐28‐h‐old amictic females were exposed to the same concentration series of juvenile hormone or precocene, precocene at 3.75 mg/L resulted in an increase in lifetime reproduction of 30.39%. However, at 0.75 and 3.75 mg/L precocene, a significantly lower percentage of mictic females was found, whereas juvenile hormone had no effect on the lifetime reproduction or mixis ratio. The population growth test showed that juvenile hormone had significant effects on the population growth rate and mixis ratio, but no effect on resting egg production. In comparison, precocene had no effect on any of these parameters. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Endogenous regulation of environmentally induced sexuality in a rotifer: a multigenerational parental effect induced by fertilisation

SUMMARY 1. Sexual reproduction in the heterogonic life cycle of many rotifers occurs when amictic females, which produce diploid eggs developing parthenogenetically into females, are environmentally induced to produce mictic females. Mictic females produce haploid eggs which develop parthenogenetically into males or, if fertilised, into resting eggs – encysted embryos which develop into amictic females after an obligatory diapause. 2. A Florida strain of Brachionus calyciflorus was used to test the prediction that amictic females hatching from resting eggs (Generation 1), and those from the next few parthenogenetic generations, have a lower propensity to produce mictic daughters in response to crowding than those from later parthenogenetic generations. In 10 replicate clones, populations initiated by amictic females from generations 1, 5, 8, 12 and 18 were exposed to a standardised crowding stimulus, and the proportion of mictic females in the populations was determined. These proportions varied significantly across generations and clones. They were very low in the early generations and gradually increased to a mean of about 0.5 at Generation 12. 3. The mechanism for the transgenerational plasticity in response to crowding is not known. One possibility is that resting eggs contain an agent from their fertilised mictic mother's yolk gland that prevents development into mictic females and is transmitted in increasingly low concentrations through successive parthenogenetic generations of amictic females. 4. This parental effect may contribute to clonal fitness by ensuring that a clone developing from a resting egg will attain a higher population size through female parthenogenesis before maximising its commitment to sexual reproduction, even in the presence of a crowding stimulus from a high population density of other clones. Therefore, the number of resting eggs to which a clone contributes its genes should be maximised. 5. The clonal variation in propensity to produce mictic females in this strain indicates genetic variation in the trade‐off between maximising population growth via female parthenogenesis and increasing the probability of producing at least some resting eggs before local extinction from the plankton.     

萼花臂尾轮虫有性生殖、种群增长和休眠卵产量间的关系

利用种群指数增长和Logistic增长模型,通过计算机模拟研究了萼花臂尾轮虫有性生殖发生的频率,后代中的混交雌体发率对种群增长和休眠卵产量的影响。在所模拟的参数范围内,随有性生殖发生频率由100％减小到20％,获得了大休眠卵产量所需的混交雌体百分率由9％增大到69％,随密度制约作用的增大（环境容纳量K值由1000减小到100）,该混交雌体百分率由18％增大到69％,休眠卵产量由1072．10降低至133．67,种群的内禀增长率与获得最大休眠卵产量所需的混交雌体百分率间呈曲线相关。当有性生殖发生得越频繁,种群增长所受的密度制约作用较小时,后代中10％－30％的个体为混交雌体时的种群中休眠卵产量较大。     

An individual-based population model for the prediction of rotifer population dynamics and resting egg production

Most species of rotifers have a combination of sexual and asexual reproduction, with sexual reproduction resulting in resting eggs, which can lay dormant for long periods. The occurrence of sexual reproduction affects population dynamics through the temporary presence of male rotifers, and a reduction in the growth of the number of female rotifers. A previously published, individual-based model used dynamic energy budget theory to describe rotifer food intake, growth, egg production, and mortality, but assumed asexual reproduction only. In the current study, we have expanded the model to describe the entire reproductive cycle of the rotifers, making it usable for investigating relationships, such as those between the signal triggering mictic egg production, and the timing and number of resting eggs produced. The model is intended for use in predicting the specific future development of cultures, for instance, as a process model in rotifer or resting egg production for aquaculture. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont & R. Rico-Martínez Advances in Rotifer Research     

Life cycle patterns of rotifers in Lake Peipsi

Life cycle strategies of rotifers in Lake Peipsi (Estonia) were examined. Bisexual (mictic) reproduction was detected in 26 species. Life cycle patterns were determined for 17 species. All three basic life cycle patterns were represented, with some evidence of intraspecific variability. The midcycle pattern, with mixis occurring near the population maximum, prevailed. Extended mixis was observed in littoral populations of Polyarthra luminosa and P. remata. Most species probably are monocyclic. Polyarthra dolichoptera and Synchaeta oblonga may be dicyclic. Mictic periods were not detected in Conochilus hippocrepis and C. unicornis, which probably are acyclic in L. Peipsi. Mictic ratios and egg ratios were calculated for some of the dominant species. The highest amictic egg ratios were observed slightly before or at the population maxima. High mictic ratios (1.0) indicated very intense bisexual reproduction in populations of A. fissa, P. dolichoptera and S. verrucosa. Definite periods of bisexual reproduction could not be distinguished in the rotifer community. During May–October, the spread of mictic reproduction merely reflects the general seasonal distribution of rotifers. Their life cycle strategies represent specific adaptations to unpredictability of their habitat. The results of the study confirm that mixis is an anticipatory event, and not a response to environment deterioration, or an ending of a rotifer population cycle.     

Genetic differentiation, behavioural reproductive isolation and mixis cues in three sibling species of monogonont rotifers

Many aquatic species usually considered to be 'cosmopolitan' have been identified as cryptic species complexes, based on deep genetic differentiation. However, reproductive isolation among sibling cryptic species has rarely been studied, and interspecific hybridization is common in some taxa.We investigated isolation mechanisms and possible introgression among three cyclical parthenogenetic rotifer species in the Epiphanes senta complex that are found in very different freshwater habitats: temperate floodplains, subtropical desert rock pools and a tropical alpine lake. Whereas Epiphanes ukera is reproductively isolated from E. chihuahuaensis and E. hawaiiensis, the latter hybridize under laboratory conditions.While reproductive isolation is incomplete, RAPD profiles indicated unique genetic signatures and showed no evidence for introgression, indicating that these three species are diverging and have independent evolutionary trajectories.Testing cues for sexual reproduction in these cyclic parthenogens demonstrated that mixis in E. chihuahuaensis and E. ukera is influenced by population density, whereas E. hawaiiensis females rarely produce mictic offspring regardless of density. Different mixis cues are likely to separate sexual periods and effectively cause reproductive isolation between the species. Epiphanes ukera and E. chihuahuaensis males display mate guarding behaviour, and E. ukera males distinguish between conspecific and heterospecific females in mate choice experiments. Geographic isolation, along with different cues for mixis induction and mate recognition, act as reproductive barriers among these sibling species.