Foundress numbers and the timing of selective events during interactions between figs and fig wasps

In intimate mutualisms between hosts and symbionts, selection can act repeatedly over the development times of the interacting individuals. Although much is now known about the overall ecological conditions that favor the evolution of mutualism, a current challenge is to understand how natural selection acts on the number and kinds of partners to shape the evolution and stability of these interactions. Using the obligate fig-fig wasp mutualism, our experiments showed that the proportion of figs developed to maturity increased quickly to 1.0 as the number of foundresses increased, regardless of whether the foundresses carried pollen. Selection against pollen-free wasps did not occur at this early stage in fig development. Within figs that developed, the proportion of galls producing adult wasps remained high as the number of pollen-carrying foundresses increases. In contrast, the proportion of galls producing adult wasps decreased as the number of pollen-free foundresses increased. Viable seed production increased as the number or proportion of pollen-carrying foundresses increased, but the average number of wasp offspring per pollen-carrying foundress was highest when she was the sole foundress. These results show that figs and their pollinator wasps differ in how fitness effects are distributed throughout the development of the interaction and depend on the number and proportion of pollen-carrying foundresses contributing to the interaction. These results suggest that temporal fluctuations in the local number and proportion of pollen-carrying wasps available to enter figs are likely to have strong but different effects on the figs and the wasps.


Foundress numbers and the timing of selective events during interactions between figs and fig wasps
Bao-Fa sun 1,2 & Rui-Wu Wang 1 In intimate mutualisms between hosts and symbionts, selection can act repeatedly over the development times of the interacting individuals. Although much is now known about the overall ecological conditions that favor the evolution of mutualism, a current challenge is to understand how natural selection acts on the number and kinds of partners to shape the evolution and stability of these interactions. Using the obligate fig-fig wasp mutualism, our experiments showed that the proportion of figs developed to maturity increased quickly to 1.0 as the number of foundresses increased, regardless of whether the foundresses carried pollen. selection against pollen-free wasps did not occur at this early stage in fig development. Within figs that developed, the proportion of galls producing adult wasps remained high as the number of pollen-carrying foundresses increases. In contrast, the proportion of galls producing adult wasps decreased as the number of pollen-free foundresses increased. Viable seed production increased as the number or proportion of pollen-carrying foundresses increased, but the average number of wasp offspring per pollen-carrying foundress was highest when she was the sole foundress. These results show that figs and their pollinator wasps differ in how fitness effects are distributed throughout the development of the interaction and depend on the number and proportion of pollen-carrying foundresses contributing to the interaction. these results suggest that temporal fluctuations in the local number and proportion of pollen-carrying wasps available to enter figs are likely to have strong but different effects on the figs and the wasps.
Mutualisms between species often involve interactions with multiple partners that may also interact with each other. At one extreme, selection could favor hosts that interact intimately with many individuals of a single clone of a vertically inherited symbiont; at the other extreme, selection could favor individuals that interact briefly with many individuals of a wide range of species, forming a multi-specific coevolving network 1,2 . The timing of selection on mutualistic partners will therefore vary with the ecological form of the mutualism. Although much is now known about the overall ecological conditions that favor the evolution of mutualism, a current challenge is to understand how natural selection acts on the number and kinds of partners to shape the evolution and stability of these interactions due to intrinsic benefit conflict between host and symbiont 3-5 . Stability in mutualistic interactions is likely to be maintained in a variety of ways at different spatial and temporal scales 1,6 as selection acts upon each component of an interaction. Hosts may evolve traits that regulate costs relative to benefits, including extreme sanctioning of partners that impose costs that exceed their benefits of the interaction 7-10 . Examples include fruit or root nodules abortion when visited only by the cheating individuals or species, as has been shown in interactions between legume and rhizobia, figs and figs wasp and yuccas and yuccas moths 5,[11][12][13][14] . Mutualisms, however, may remain stable even through more subtle means, as hosts evolve, for example, to restrict the activities of partners in ways that are less detrimental to host fitness. Examples include restriction in the length of time that a host makes a reward available or restriction of access to the reward itself, as occurs in production of floral rewards by plants [15][16][17][18] . In some cases, however, stability may be the result of a combination of selection pressures on hosts and partners that happen to result in mutualism as selection favors participants on each side of the interaction that maximize their own fitness 5,19 . The interactions between plants and pollinating floral parasites provide opportunities to evaluate how fitness in each participant is built up throughout the course of an interaction from the time of encounter to the production of seeds and the next generation of adult pollinators. These interactions occupy a complex middle ground between intimate symbiotic mutualisms and mutualisms among free-living species. More than a thousand plant species worldwide are involved in these mutualisms, which have originated repeatedly among plant families 8,[20][21][22][23] . The interaction begins as one or more pollen-carrying adult insects lay eggs into the flowers while simultaneously pollinating the flowers 20 . Thereafter the fitness of the developing host plant and that of the developing larvae of pollinators are fully linked. The interaction proceeds through several stages, each of which provides an opportunity for selection to act on the partners.
The interactions between figs and fig wasps are among the most well-known and diverse mutualisms found in nature, and they have persisted for millions of years 24,25 . In these interactions, one or more pollen-

Materials and Methods study site. The study was carried out in and near the Xishuangbanna Tropical Botanic Garden (XTBG) in
Yunnan province, China (21°41′N, 101°25′E). This garden is approximately 600 m above sea level and has a monsoonal climate. The rainy and dry seasons last from May to October and from November to April, respectively. Mean annual precipitation is 1,557 mm with about 80% occurring during the rainy season 30 . The mean annual temperature is 21 °C, and the mean annual relative humidity is 87%. All experiments were performed between May and September of 2011. study species. Ficus racemosa is widely distributed from India to Australia 31 and resides in the Sycomorus section of Ficus. Ficus racemosa is a monoecious fig and it is actively pollinated by Ceratosolen fusciceps 31 . Ficus racemosa is usually a large free-standing tree (up to 25 m in height). It produces large crops of large spherical figs (up to approximately 45 mm in diameter when mature), which grow on short branches (racemes) that are attached to the trunk and larger branches. The flowering patterns of F. racemosa are typical for monoecious Ficus, with intra-tree synchrony and inter-tree asynchrony 21 . Both wasps and seeds are produced in each fig resulting in a clear conflict between the mutualists as during seed and larval development, especially when the unutilized common resource is limited as a result of foundresses increase 32 . Seed production, however, is only one component of plant fitness. In order to reproduce through spread of both female and male gametes, a tree must produce seeds and also wasps for pollen dispersal. In contrast, wasps only need to produce more wasps and they do so by galling some host flowers.

Methods
We used Ficus racemosa and its pollinator wasps Ceratosolen fusciceps to assess the effects on fitness of the number of wasps carrying pollen relative to number not carrying pollen. The experiments used two categories of wasps. Pollinating wasps carried pollen to the figs in pollen pockets that they had collected naturally as they emerged from their natal figs; pollen-free wasps were prevented from carrying pollen by collecting specimens from treated syconia in which we removed the stamen before the adult wasp emerged from the galled flowers. This manipulation of syconia to prevent wasps from collecting pollen has been successfully used in previously studies 5,10 .
In one set of experiments, we introduced either pollen-free (P−) or pollen-carrying (P+) wasps into receptive fruits. In these experiments, we conducted five treatments in which either 1, 3, 5, 7 or 9 foundresses were introduced. The foundresses were sequentially introduced into each receptive syconium within two days, allowing a 2 hour time interval between introductions of each wasp when the total foundress number was high. Some data from these experiments has been used in our previous paper that addressed a different set of questions 10,18 . In another set of experiments, we introduced 9 wasps, but the number of pollen-carrying wasps within that total varied from 0, 2, 4, 6, 8 to 9. The sample size of each treatment is larger than 20. Collectively, these experiments allowed an evaluation of a wide range of ecologically and evolutionarily relevant patterns of colonization of figs by fig wasp foundresses and the responses of figs under all these scenarios.
In all experiments, we selected 2-4 trees from two sample sites of locally fragmented and highly fragmented forest. When the treated syconia developed to maturity, but before the pollinator wasps cut exit holes, we collected the syconia and injected a solution of 75% ethanol, killing the present adult wasps. We opened each synconium, cut open each gall, and counted the viable seeds, and male wasps, and female wasps. We also cut open each gall to ensure that the results also included any well-developed adult wasps that had not yet emerged from their natal gall. Mature syconia had empty galls that contained no adult wasps. Such galls may result when females successfully oviposited but larvae failed to develop 10,12 . Statistics analysis: SPSS 17.0 was used to conduct all the statistics. The fig size is an independent parameter and therefore is included as covariate. The sample site (fragmented or forested) and tree are also included as a covariate and GLM or one way ANOVA are used in regression analysis and mean comparison analysis. We use Pearson correlation in the correlation analysis.   (Fig. 1B). This result suggests that there was little interference between wasps during this stage of the interaction. Within figs that developed, the proportion of galls producing adult wasps remained high as the number of pollinating foundresses increased significantly (N = 101, r = 0.30, P < 0.01) (Fig. 1C). In contrast, the proportion of galls producing adult wasps decreased significantly as the number of pollen-free foundresses increased (N = 101, r = 0.92, P < 0.001). This, then, was the first developmental stage at which selection acted against P− foundresses.

Results
The total number of adult wasps produced by a fig increased significantly as the number of pollinating foundresses increased up to seven wasps (N = 101, r = 0.73, P < 0.001) (Fig. 1D). This pattern may result from the greater number of seeds in figs founded by multiple P + wasps (Oneway ANOVA  (Fig. 1E). In figs entered by more than one wasp, the number of wasps produced per foundress was similar for 3-9 wasps for P + wasps (n = 80, r = 0.164, P < 0.001) but decreased linearly for P− wasps (n = 81, r = 0.95, P < 0.001). However, when only visited by one foundress, the fig develop similar proportion galls containing the adult wasps regardless whether the foundress carry the pollen or not (t = 1.06, df = 38, P = 0.30). Hence, selection favors wasps that are single foundresses and acts most strongly against pollen-free wasps that enter figs with many other pollen-free wasps.  (Fig. 2C,B). Hence, selection would act most strongly against P− fig wasps that entered a fig with many other P− fig wasps and induced many galls. The same pattern was evident for the number of adult wasps produced per fig (Fig. 2C). That result is reinforced if the values are scaled to the number of adult offspring wasps produced on average per foundress wasp, but pollinating wasps also suffered lower offspring production in figs with many galls (Fig. 2D).
Overall, the sequence of fig and wasp developmental stages, mediated by the number of foundresses and the number of galls, resulted in a marked difference in the number of adult wasps produced by figs containing pollinating wasps as compared with figs containing pollen-free wasps. Pollen-carrying and pollen-free foundresses produced most offspring per foundress when they entered syconia alone. Pollen-free foundress wasps, however, suffered consistently lower production of offspring at all levels of foundress numbers or gall numbers.  (Fig. 3B,E).
The effects of the number of foundress wasps on seed production differed from the effects on wasp production. Figs with nine foundress wasps produced the same number of galls, regardless of the combination of P + and P− foundresses entering the fig (F 5, 145 = 0.28, P = 0.95), (Fig. 3F). Figs with nine pollen-free wasp foundresses produced no seeds, demonstrating that all pollen had been removed in the P− syconia treatment (Fig. 3F). As the ratio of P− to P+ wasps increased from 1:8 to 9:0, however, the number of seeds produced increased linearly (y = 81.744 + 204.129 × , y = number of seeds, x = number of pollen-carried wasps, Linear:R 2 = 0.959, F = 3491.27, df 1,149 , P < 0.001). Hence, from the perspective of plant fitness, more pollinating foundresses are better than few foundresses, at least up to nine foundresses.

Discussion
The  (Fig. 1E). In this situation, there is low overall production of galls which may benefit development of the offspring (Fig. 2D). This result might explain why the foundresses with stronger competitive ability are selected for in the process of evolution, because the strong interference among the foundresses 4,33 and even fight and kill among the foundresses 34 will decrease the overall egg deposition of foundresses. The interference, though, is asymptotic. Some direct or indirect interference may occur among small numbers of pollinating foundresses, as shown in the lower number of offspring produced per female per fig in multi-foundress figs. The effects, however, do not continue to amplify as the number of foundresses increases in these experiments (Fig. 1E). In contrast, P− foundresses suffer decreased fitness with each additional P− foundress. This result may occur either through selection that has favored direct sanctioning of figs that have many P− foundresses, or indirectly through physiological mechanisms that withdraw resources from figs with many galls but few developing seeds 12,35 . Pollen-free foundresses may fare best when entering figs with a small number of pollen-carrying foundresses. The mutualism is therefore regulated in part through the effects on the number of wasps produced relative to the number and ratio of P+ and P− foundresses.
In  33 . Such mechanism could prevent the potential conflict between the figs seed production and pollinator offspring production, because both seed production and wasp offspring must be at the expense of common resource-female flowers of figs 4,36-38 . Selection on figs could therefore act to optimize the number of foundresses that can enter either simultaneously or sequentially 33,39 .
In The foundress number usually is between 7-9 foundresses in the study population, based on the results of our experiments and observations. The interference competition among foundresses, if foundress number is higher than 9, might lead to a decrease of production of galls and seeds 4,33 .
When P+ and P− wasps enter fig cavities together, the fitness effects differ on host figs and the pollinating wasps. Selection on figs should select for more pollinating foundresses. Selection on figs should also favor plants that can distinguish galls produced by pollen-carrying foundresses from those not carrying pollen 11 . In the absence of such targeted sanction by figs. 11 , selection may favor wasps that do not carry pollen if pollen collection and dispersal is costly to fitness.
The combination of antagonistic and mutualistic selection between the host and symbiont might lead to population oscillations among the host, pollinating wasps and cheating wasps or parasitic wasps 5,10,41 . Moreover, evolutionary changes in the ability to distinguish mutualists from cheaters may explain shifts in the evolution and prevalence of parasites and mutualists over the evolutionary time scale in these kinds of interaction 5,42,43 . It may also partly explain the coexistence of parasitic wasps (including the cheating individuals of pollinating wasps and non-pollinating wasps) and cooperative individuals in some plant populations but not necessarily in all populations 13,[43][44][45][46] .
The results also suggest that figs, P+ wasps, and P− wasps may all differ in the stages of the interaction at which selection acts mostly strongly. As a result selection may favor figs that are able to respond to the number and ratio of P+ and P− wasps entering a fig. Fig trees abort the fruits in which non-pollinating wasps Sycophaga testacea oviposit before pollinating wasps 5,29 , and fig trees often also abort fruits only entered by only one foundress. This abortion may be a pre-adaptation mechanism that could save the resource for the fig trees 47,48 . However, fig trees often do not abort fruits in which pollen-free wasps of Sycophaga mayri oviposit later than pollinating wasps. Even the female offspring of pollen-free pollinating wasps can contribute to fig fitness because, once they emerge, they could disperse pollen to another tree.
Fig wasps may also diversify their behaviors to maximize their fitness. Selection on wasps could act to separate emergence times and oviposition peaks among individuals within and among species to avoid the competition 5,49-51 . The results reported here also suggest that pollinator wasps might diversify their strategies at the early stages. Even after entering the fig cavities, selection might act on foundresses to avoid ovipositing at the same time, if interference or fruit abortion occurs above a threshold of simultaneous oviposition attempts.

Conclusion
The experiments here showed that figs and their pollinator wasps differ in how fitness effects are distributed throughout the development of the interaction and depend on the number and proportion of pollen-carrying foundresses contributing to the interaction. The results suggest that selection on the partners may differ in intensity during different stages of a mutualistic interaction. Hence, stability of mutualisms may sometimes be maintained under some ecological conditions, even without direct selection on hosts to sanction partners, through the combined selection pressures acting on each species across the developmental stages of the participants.

Data Availability
If the paper get published, the data could be available through the online of the journal.