Effects of sexual reproduction of the inferior competitor Brachionus calycifl orus on its fitness against Brachionus angularis *

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1 Chinese Journal of Oceanology and Limnology Vol. 33 No. 2, P , Effects of sexual reproduction of the inferior competitor Brachionus calycifl orus on its fitness against Brachionus angularis * LI Chen ( 李陈 ), NIU Cuijuan ( 牛翠娟 ) ** Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, College of Life Sciences, Beijing Normal University, Beijing 1875, China Received Mar. 2, 214; accepted in principle Jun. 9, 214; accepted for publication Aug. 18, 214 Chinese Society for Oceanology and Limnology, Science Press, and Springer-Verlag Berlin Heidelberg 215 Abstract Sexual reproduction adversely affects the population growth of cyclic parthenogenetic animals. The density-dependent sexual reproduction of a superior competitor could mediate the coexistence. However, the cost of sex may make the inferior competitor more vulnerable. To investigate the effect of sexual reproduction on the inferior competitor, we experimentally paired the competition of one Brachionus angularis clone against three Brachionus calycifl orus clones. One of the B. calycifl orus clones showed a low propensity for sexual reproduction, while the other two showed high propensities. The results show that all B. calycifl orus clones were excluded in the competition for resources at low food level. The increased food level promoted the competition persistence, but the clones did not show a clear pattern. Both the cumulative population density and resting egg production increased with the food level. The cumulative population density decreased with the mixis investment, while the resting egg production increased with the mixis investment. A trade-off between the population growth and sexual reproduction was observed in this research. The results indicate that although higher mixis investment resulted in a lower population density, it would not determinately accelerate the exclusion process of the inferior competitor. On the contrary, higher mixis investment promoted resting egg production before being excluded and thus promised a longterm benefit. In conclusion, our results suggest that mixis investment, to some extent, favored the excluded inferior competitor under fierce competition or some other adverse conditions. Keyword : sexual reproduction; competition; resting egg; Brachionus 1 INTRODUCTION Cyclic parthenogenetic rotifers could reproduce sexually or asexually and take advantage of the best of both reproductive modes, just like other parthenogenetic animals (Simon et al., 22). Therefore, they are adequate model animals for assessing the role of sex in species competition. Sexual reproduction may dampen current population growth and competition efficiency (Serra and King, 1999), as higher energy is required for a fertilized mictic female to lay a resting egg than for an amictic female to produce a daughter (Gilbert, 21). Furthermore, clones with a high propensity for sexual reproduction are restricted to significantly lower equilibrium population sizes (Stelzer, 212). The density-dependent cost of sex makes it possible to mediate species coexistence apart from food partitioning (Ciros-Pérez et al., 21) and predation (Ciros-Pérez et al., 24; Yin and Niu, 28). Zhang and Hanski (1998) demonstrated that negative feedback from features of sexual reproduction, such as the density-dependent sex ratio, sexual conflict and sexually transmitted diseases, could promote species coexistence without separate niches. Montero-Pau and Serra (211) showed that a density-dependent life cycle switch, such as the asexual to sexual transition of the high-density competitor, could promote the coexistence of species without niche differentiation. * Supported by the National Natural Science Foundation of China (No ) ** Corresponding author: cjniu@bnu.edu.cn

2 No.2 LI and NIU: Effects of sexual reproduction on the inferior competitor 357 Theory required that the density-dependent sexual reproduction or diapause investment may be controlled by species-specific signals. By investing in sexual reproduction, a high-density competitor would decrease its own population growth rate more than that of its low-density competitor, allowing the latter to grow faster and have the potential to coexist (Montero-Pau and Serra, 211). However, according to the theory, investment to the sexual reproduction, which is also called mixis investment in parthenogenetic rotifers, might make the inferior competitor more vulnerable. Competition between sympatric cryptic Brachionus plicatilis species has proven that the exclusion of the inferior competitor was related to higher investment in sexual reproduction (Ciros-Pérez et al., 22). These cryptic species were found to share the same crowding signal (García-Roger et al., 29), which accelerated the exclusion of the inferior as the result of earlier and higher sexual reproduction. However, during interspecific competition, the result might be different, as sex is separately density dependent (Gilbert, 1963). During sexual reproduction, fertilized mictic females produce resting eggs, that are dormant for some time before hatching. They are responsible for the initiation of the rotifer population after periods of adverse conditions, and they are also a means of dispersal (Gilbert, 1974; Pourriot and Snell, 1983). The formation of resting eggs, which consists of a bank, could buffer against stochasticity and has been suggested as a type of time dispersal that somehow mediates the long-term coexistence of competitors (Chesson, 2). Hence, the production of resting eggs could be regarded as a feature of the long-term fitness. B. angularis and B. calycifl orus are two common rotifer species in freshwater lakes and ponds. The former is smaller than the latter and has a lower threshold food level (TFL) (Stemberger and Gilbert, 1987), which promotes its ability to survive and grow at lower food concentrations. According to the resource competition theory (Tilman, 1982), B. angularis would outcompete, or even exclude, the inferior competitor B. calycifl orus when food resources are limited. B. calycifl orus clones with high sexual reproduction propensity are frequently found, which implies that mixis investment may benefit fitness. For example, B. calycifl orus could have produced enough resting eggs before being excluded in the growing season to establish a new population in the following growing seasons. Therefore, the inferior competitor avoids being excluded via mixis investment during the competition. Thus, clones with a high sexual reproduction propensity would be selected during the short growing season, if higher mixis investment results in more resting eggs. Based on the above assumption, we hypothesized that the inferior competitor may be affected by sexual reproduction in at least two aspects during interspecific competition: (1) the allocation of resources in resting eggs may decrease the competitive capabilities of the active population and accelerate its exclusion in the short term, (2) while high investment in sexual reproduction may result in more resting eggs and thus promote long-term benefits. In this paper, we tested the hypothesis by analyzing the relationship between sexual reproduction and the short-term or long-term performance of the inferior competitor B. calyciflorus in interspecific competition with B. angularis at low and high food levels. The results may provide evidence to explain the selection of high mixis investment clones in the evolution process. 2 MATERIAL AND METHOD 2.1 Culture conditions of food algae and rotifers The green algae Chlorella pyrenoidosa was used as the food for rotifers in this research. They were cultured in SE medium (FACHB-Collection, 214) at 25 C with continuous illumination (3 lx), separated from the culture medium by centrifuging for 5 min at 7 r/min, and suspended in modified MBL medium (Stemberger, 1981). The concentrated algae were stored at 4 C in the dark, and their concentration was determined using a blood cell counting panel. The rotifers B. angularis and B. calycifl orus were isolated from a natural lake in Houhai Park, Beijing, China ( E, N). Each clone was established from a single parthenogenic female and maintained in MBL medium, and three clones of the rotifer B. calycifl orus (,, ), which differed in their propensity for sexual reproduction, and one clone of rotifer B. angularis, were then chosen for the experiments. All rotifers in the experiments were maintained at 2±.5 C in a 16:8 h L:D photoperiod in a growth chamber. The sexual reproduction propensity of B. calycifl orus at a density of 1 ind./ml was determined following Gilbert s protocol (Gilbert and Schröder, 27):, 11% (19 neonates);, 45% (19 neonates);, 46% (192 neonates). Preculture populations were kept at a low population density (<1 ind./ml) with cells/ml C.

3 358 CHIN. J. OCEANOL. LIMNOL., 33(2), 215 Vol.33 pyrenoidosa for at least three months prior to the experiments. 2.2 Experimental design The competition experiment between B. angularis and B. calycifl orus was conducted in a 5-mL transparent beaker containing 2 ml MBL medium. Two food levels were set: cells/ml (high food level) and cells/ml (low food level). The experiment was initiated with 4 B. angularis and 1 B. calycifl orus individuals (randomly chosen from pre-culture populations). In total, this experiment consisted of 24 groups (two food concentrations, three B. calycifl orus clones and four replicates). All rotifers were transferred to 2 ml fresh medium daily with algae of the corresponding concentration, and densities of Brachionus species were assessed. The individuals of B. calycifl orus were exhaustively counted and classified as amictic females, nonovigerous females and mictic females. The numbers of well-shaped resting eggs that B. calycifl orus produced were recorded. Total counts were initially taken for B. angularis. When the number subsequently reached 4 individuals in one beaker, three samples of 2 ml medium were obtained to estimate the number of B. angularis. The counted rotifers were transferred back to the population. The experiment was terminated when the trends of most populations became obvious and stable. 2.3 Data analysis The mixis ratios were calculated as the proportion of egg-bearing mictic females, and the integrated mixis ratios was referred as the mixis investment (Ciros-Pérez et al., 22). The cumulative population density, which was calculated by integrating the population density over time, was defined as an indicator of the species ability to seize the niche in a growing season. The total number of well-shaped resting eggs that each clone produced was calculated. Population growth rate ( r ) was obtained with the equation: r =(lnn t lnn )/t, where N and N t are the initial and peak population densities, t is the duration time in days (Yin and Niu, 28). A spearman correlation analysis was conducted to estimate the relationship between resting egg production, cumulative population density, and mixis investment. The population growth dynamics and the mixis ratio curves were compared using repeated measures ANOVA, after verifying the parametric assumptions of normality (K-S test), homoscedasticity (Levene test) and sphericity (Mauchly test). The Mauchly's test result showed that the assumption of sphericity was not satisfied ( P <.5), so Greenhouse- Geisser correction for df was used here. The cumulative population density, mixis investment, and resting egg production were compared with one-way ANOVA and a Tukey tests for multiple comparisons. 3 RESULT The population dynamics of B. angularis and B. calycifl orus clones and the time course of mictic ratios of B. calycifl orus are shown in Fig.1 (low food level) and Fig.2 (high food level). Irrespective of the food level, B. angularis required more time to reach a much higher maximum population density than B. calycifl orus. At the low food level, B. angularis outcompeted all three B. calycifl orus clones. When population density did not significantly differ from in 3 continuous days, the population was thought to be excluded. Clones and were excluded on day 11. Clone, which maintained the lowest population density throughout the experiment, was the last clone to be excluded on day 14, and B. angularis required more time to reach the maximum population density when competing with clone (Fig.1). The maximum population density of B. angularis was higher and occurred later. Clone, which showed low mixis ratios throughout the population growth, reached a maximum population density that was approximately twice of that at low food level. Clone, which showed high mixis ratios, maintained a small population until the end of the experiment. Clone was eventually excluded. All B. calyciflorus clones tended to persist with B. angularis for a much longer time at high food level (Fig.2). Repeated measures ANOVA test show that clone and food level have significant effect on the population growth dynamics (df=2.5, F= 14.1; df=4.1, F =29.7). The mixis investment of B. calycifl orus at low food level was,.157;,.398; and,.212. At high food levels, these values were,.66;,.333; and,.384 (Fig.3). Tukey test showed that the mixis investments of and were significantly higher than that of ( P <.5), except for at a low food level ( P =.198). The result of the repeated measures ANOVA showed that the mixis ratios of B. calycifl orus were significantly different ( P <.5) among clones. The cumulative population density of the B. calycifl orus clones and were significantly smaller than that of at both low (, P =.1;,

4 No.2 LI and NIU: Effects of sexual reproduction on the inferior competitor 359 Population dentsity (ind./ml) Time (d) BA Mixis ratio Time (d) Fig.1 Population dynamics of Brachionus calyciflorus and Brachionus angularis (left panels) and mixis ratios of B. calyciflorus (right panels) at cells/ml of Chlorella pyrenoidosa BA= B. angularis, which was cultured with each rotifer B. calycifl orus clone populations (,, and ). Different vertical axes were used here for B. calycifl orus (left vertical axis) and B. angularis (right vertical axis) as population density of B. angularis was much higher than that of B. calycifl orus. Data are mean±standard error values. Population dentsity (ind./ml) Time (d) BA Mixis ratio Time (d) Fig.2 Population dynamics of B. calyciflorus and B. angularis (left panels) and mixis ratio of B. calyciflorus (right panels) at cells/ml of C. pyrenoidosa BA= B. angularis, which was cultured with each rotifer B. calycifl orus clone populations (,, and ). Different vertical axes were used here for B. calycifl orus (left vertical axis) and B. angularis (right vertical axis) as population density of B. angularis was much higher than that of B. calycifl orus. Data are mean±standard error values.

5 36 CHIN. J. OCEANOL. LIMNOL., 33(2), 215 Vol Low food level High food level 5 Low food level High food level Mixis investment Number of resting eggs Clone Fig.3 Mixis investment of different B. calyciflorus clones (,, ) at cells/ml and cells/ml C. pyrenoidosa. Data are mean±standard error values Asterisks indicate significant differences relative to at each food level. * P <.5, ** P <.1. Clone Fig.5 Resting egg production of different B. calyciflorus clones (,, and ) Data are mean±standard error values. Asterisks indicate significant differences relative to at each food level. * P < Low food level 5 -H Cumulative population density (ind./ml) Clone High food level Fig.4 Cumulative population density of different B. calyciflorus clones (,, and ) at cells/ml and cells/ml C. pyrenoidosa Data are mean±standard error values. Asterisks indicate significant differences relative to at each food level.* P <.5. P =.5) and high (, P =.1;, P =.2) food levels (Fig.4). In addition, the difference grew as food level increased (1.88- and 2.17-fold of and at low food level, and 2.52-fold at high food level). The resting egg production of different rotifer B. calycifl orus clones is shown in Fig.5. Irrespective of the food concentration, clone produced fewer resting eggs than the other two clones. All clones tended to produce more resting eggs when food was abundant. Clones and produced significantly ( P <.5) more resting eggs than clone at low and high food levels, but this difference was not significant for clone at a low food level ( P =.729). The cumulative population density decreased with Number of resting eggs L 2 -L -H -L -H Cumulative population density (ind./ml) Fig.6 Relationship between resting egg production and cumulative population density of B. calyciflorus -H and -L represent high (3 1 6 cells/ml) and low (1 1 6 cells/ ml) food level, respectively. Data are mean±standard error for each clone. the mixis investment and the resting egg production increased with the mixis investment. But no significant correlation was detected using spearman test (cumulative population density, R =-.429, n =6, P =.397; resting egg production, R =.657, n =6, P =.156). For a given clone, the cumulative population density increased with food level and the resting egg production increased with the cumulative population density (Fig.6). The cumulative population density, resting egg production, and population growth significantly differed among clones and were significantly affected by the food level (Table 1). Mixis investment varied significantly among clones but was not affected by the food level.

6 No.2 LI and NIU: Effects of sexual reproduction on the inferior competitor 361 Table 1 Two-way ANOVA analysis on different variables of Brachionus calyciflorus population Source df SS MS F P Cumulative population density Clone (A) <.1 Food level (B) <.1 Interaction A B <.1 Error Mixis investment Clone (A) <.1 Food level (B) Interaction A B <.1 Error Resting eggs Clone (A) <.1 Food level (B) <.1 Interaction A B Error Population growth rate Clone (A) <.1 Food level (B) <.1 Interaction A B Error DISCUSSION A trade-off between sexual reproduction and asexual reproduction was observed in this study (Fig. 6). A species investing less in sexual reproduction become a better competitor, which might be compromising its own long-term persistence, while a species with a high investment in sexual reproduction gain less potential for population growth via female parthenogenesis, and thus a low population density. In this study, the and clones, which both possessed a high sexual reproduction propensity, had a significantly lower cumulative population density than, a low mixis investment clone (Fig.4). A similar trade-off between the sexual ratio and carrying capacity has been described in B. plicatilis (Dimas- Flores et al., 213). A low population density would reduce the ability of the rotifer to offset predation loss (Gilbert and Dieguez, 21) and increase the possibility of extinction due to random walks of the environment (Montero-Pau and Serra, 211). Furthermore, a low population density may result in a low probability of encounters between males and young mictic females capable of being fertilized, and thus a limited production of resting eggs overall, as demonstrated by the performance of the clone at a low food level. Therefore, mixis investment likely reduced the short-term fitness of the clone. However, more investment in sexual reproduction may result in more resting eggs, and this increase ensures a long-term benefit for the species (Chesson, 2). Our results showed that and, which had a high propensity for sexual reproduction and lower population density, produced more resting eggs than clone during the experiment (Fig.5). When the growing season is short, especially when the risk from invertebrate predators and competitors is high, a very low mixis threshold and high maximal mixis ratio may ensure the production of some resting eggs soon after the colonization of the pond and before the complete removal from the plankton (Schröder et al., 27; Gilbert and Dieguez, 21). Initiating sexual reproduction shortly after the population emerges from resting eggs and hence at a low population density, the rotifers could produce some resting eggs for the sediment egg bank before being excluded or consumed by predators. All of these behaviors promise high mixis investment clones a long-term advantage during short growing season. What s more, sometimes mixis investment does not determinately reduce the short-term competitive ability of the inferior competitor, and various mechanisms could reduce the risks due to a low population density. The persist time of B. angularis did not show an obvious pattern, for either and (with more mixis investment) or (with less mixis investment). A low population density may have increased the possibility of random exclusion. Furthermore, both clones initiated at low densities showed a decreased population growth rate in competition due to high investment, would slow the depletion of food resources during the early period and might delay the resource competition. Moreover, sometimes a low population density does not result in a small population for a clone. On the contrary, a low population density may imply a large population size in a large water pond, and individuals may aggregate and experience local densities much higher than the mean because of responses to light, food or predators (Gilbert, 24). Furthermore, certain behaviors may facilitate male-female encounters in low-density natural populations; the frequency of encounters could increase as the swimming speeds of males, females or both increases. Furthermore, an increase in the reproductive rate in the presence of predators can

7 362 CHIN. J. OCEANOL. LIMNOL., 33(2), 215 Vol.33 have a stronger adverse effect because an increase in the food availability will also increase the predator density, which further reduces the abundance of prey (Dumont et al., 1995). The results of the resource competition between rotifers are mainly decided by the threshold food level (TFL) for reproduction (Stemberger and Gilbert, 1985), and the rate of competitive exclusion has been suggested to decrease as the difference between the TFL of the two species decreases (Grover, 1997). Our result is consistent with this prediction, and as an inferior competitor with a higher TFL, all B. calycifl orus clones were eventually excluded by B. angularis in the competition experiments that had a lower TFL for reproduction. Our results also show that an increased food level supply slowed the competitive exclusion but did not change the competitive outcome or allow coexistence. At a high food level, all B. calycifl orus clones persisted with B. angularis for a much longer time. Clone, which had a low investment in sex, could grow to a higher population density with more resting egg production at a high food level. This finding was consistent with the observation that low investment sex was favored during the growing season when food resources were abundant (Carmona et al., 29). Because both the mixis ratio and population density decide resting egg production, a minimal population density is required to produce resting eggs because few resting eggs would be formed if the population density is too low, as was observed for at a low food level in this study (Fig.5). Data from field experiments (Schröder and Gilbert, 24; Serra et al., 25) have proven that sexual reproduction from the resting eggs is significantly delayed in early generations of most species from temporary ponds, which indicates an increase in abundance for some generations via parthenogenesis before committing to sexual reproduction. Thus, rapid population growth was initially favored and later maximized resting egg production, which is another mechanism that favors sexual reproduction. Because the population growth rate of a species was not a good indicator of competitive ability (Sarma et al., 1999), we ignored this parameter here. However, we observed an interesting fact: B. angularis (BA) grew significantly slower while competing with than that with and at a low food level. This finding may explain why persisted with BA slightly longer than and did at a low food level. Further studies are needed to determine whether B. calycifl orus would inhibit the population growth of B. angularis via chemical signals or other mechanisms. In conclusion, our results show that the mixis investment of the inferior competitor may not accelerate the process of its exclusion during interspecific competition under certain circumstances, although it results in a low maximum population density. Moreover, additional resting egg production due to high sexual reproduction promises long-term benefits for the inferior competitor. However, the fitness of the species always experiences a trade-off between the asexual reproduction and sexual reproduction in a given environment. 5 ACKNOWLEDGEMENT We are grateful to SUN Dong and ZHANG Zuobing for their valuable comments on the manuscript and polishing of the English. References Carmona M, Dimas-Flores N, García Roger E, Serra M. 29. Selection of low investment in sex in a cyclically parthenogenetic rotifer. Journal of Evolutionary Biology, 22 (1): Chesson P. 2. Mechanisms of maintenance of species diversity. Annual review of Ecology and Systematics, 31 : Ciros-Pérez J, Carmona M J, Lapesa S, Serra M. 24. Predation as a factor mediating resource competition among rotifer sibling species. Limnology and Oceanography, 49 (1): 4-5. Ciros-Pérez J, Carmona M J, Serra M. 21. Resource competition between sympatric sibling rotifer species. Limnology and Oceanography, 46 (6): Ciros-Pérez J, Carmona M J, Serra M. 22. Resource competition and patterns of sexual reproduction in sympatric sibling rotifer species. Oecologia, 131 (1): Dimas-Flores N, Serra M, Carmona M J Does genetic diversity reduce intraspecific competition in rotifer populations? Hydrobiologia, 75 (1): Dumont H J, Sarma S S S, Ali A J Laboratory studies on the population dynamics of Anuraeopsis fi ssa (Rotifera) in relation to food density. Freshwater Biology, 33 (1): FACHB-Collection SE (Brostol s solution) from Freshwater Algae Culture Collection of the Institute of Hydrobiology. aspx?product=3. Accessed on García-Roger E M, Dias N, Carmona M J, Serra M. 29. Crossed induction of sex in sympatric congeneric rotifer populations. Limnology and Oceanography, 54 (6):

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