Acta Ecologica Sinica 32 (2012) 99–103

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Acta Ecologica Sinica

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Spatial distribution patterns of three fig wasps on Ficus semicordata: Hownon-pollinators affect pollinator’s sex ratio

Xiao Yan a,b, Yan-Qiong Peng a, Da-Rong Yang a,⇑a Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming 650223, Chinab Graduate University of Chinese Academy of Sciences, Beijing 100049, China

a r t i c l e i n f o

Keywords:Ceratosolen gravelyi

Pollinator fig waspNon-pollinating fig waspsSex ratioSpatial distribution

1872-2032/$ - see front matter � 2012 Ecological Socdoi:10.1016/j.chnaes.2012.02.003

⇑ Corresponding author.E-mail address: yangdr@xtbg.ac.cn (D.-R. Yang).

a b s t r a c t

Sex ratio theory is one of the most productive fields in research on evolutionary biology. Pollinating figwasps, due to their particular natural life history, are considered to be a valuable model for the studyof sex ratio evolution. A great deal of research concerning the factors that affect pollinator fig wasp (Aga-onidae) progeny sex ratio has been done, and at present three main factors (haplodiploidy, local matecompetition and inbreeding) are found to be important at the population level. However, there still existsvariation between empirical data and model predictions. Another factor to which little thought has beengiven before is the effect of non-pollinating fig wasps (NPFWs) which parasitize in the larvae gall of pol-linator thus kill pollinators and exploit the fig/fig pollinator mutualistic systems. In this study, we focuson why and how non-pollinating fig wasps distort pollinator fig wasp’s original sex ratio. Through con-trolling the number of ovipositing foundresses inside a fig, combined with the observation of ovipositingbehavior and sequence, we studied three species of wasp in the figs of a dioecious fig Ficus semicordataincluding the pollinator Ceratosolen gravely and NPFWs Platyneura cunia, Sycoscapter trifemmensis in trop-ical area of Xishuangbanna from September to December 2009. First, we observed the timing of oviposi-tion of all fig wasps utilizing F. semicordata and found differences when compared to previous studies.Such as P. cunia is the fourth rather then the secondary fig wasps to oviposit on the syconia approximately10 days after the pollinator. S. trifemmensis oviposits much earlier than previously thought, 14–32 daysafter the pollinators. We examined the spatial location of male and female progeny of the pollinator.We found foundresses of pollinator prefer to use innermost ovules first. Only at high offspring numberswere the outer ovules used. More male pollinator offspring were developed near the fig cavity, whilefemale pollinator offspring were more evenly distributed among ovule layers. As pollinator offspringnumbers increased, this phenomenon became more pronounced. This pattern of segregation of male lar-vae gall in inner ovules and female larvae gall in outer ovules suggests that female offspring might bemore vulnerable to attack by parasitic wasps that oviposit from outside the syconium. Experiments laterdemonstrated that NPFWs are restricted by their ovipositor length and they prefer to or can only lay theireggs into ovules near the fig wall. Then we examined the spatial location of NPFWs and compared thiswith the spatial location of male/female progeny of pollinator. NPFWs had a high probability of parasit-izing female pollinator larvae. Thus, NPFWs have a substantial effect on the sex ratio of the pollinator, asparasitism risk decreases towards the center of the syconium, where inner ovules provide enemy-freespace for most of male pollinator offspring. Partial correlation analyse shows that sex ratio of pollinatorprogeny has a positive relationship with the number of NPFWs. We suggest that the resulting gradient inoffspring viability between male and female contributes to selection on pollinators’ for a less female-biased sex ratio. When the affect of NPFWs was excluded, the pollinator sex ratio was not in good agree-ment with local mate competition theory, although it was still female-biased. In addition, the averagenumber of offspring per foundress decreased with increasing foundress number, but pollinator sex ratiowas positively related to brood size. Thus, pollinator females do not appear to adjust their sex ratio tofoundress density directly, but use brood size and foundress density simultaneously as cues to assesspotential LMC.

� 2012 Ecological Society of China. Published by Elsevier B.V.

iety of China. Published by Elsevier B.V.

100 X. Yan et al. / Acta Ecologica Sinica 32 (2012) 99–103

1. Introduction

Sex allocation strategy is closely related to fitness. And sex ratiois one of the productive areas in the research of evolutionary biol-ogy. Fig wasps, due to their particular natural life history of fig-pol-linating, can only mate and reproduce in a small population which isestablished by few foundress. This kind of natural life history allowsthe testing and refinement of theory that concerns local mate com-petition and inbreeding and their combined effects on sex allocationand mating system. Consequently, fig wasps are considered to be aperfect research material for the study of sex ratio evolution [1–5].

At present, three main factors are found to be quite importantto pollinator fig wasp (Agaonidae)’s progeny sex ratio at the popu-lation level, that are haplodiloid, LMC and inbreeding. Haplodiloidmakes parthenogenesis, and that means unfecundated eggs willdevelop to haploidic male while fecundated eggs will develop todiploidic female. These cause genetic relationship changes be-tween different sexes and generations, and the results is foundressadjust the sex ratio of their offspring by controling the fecundation.LMC predicts female-biased sex ratio. Herre uses foundress num-ber to estimate the intensity of LMC, and found in many pollinatorfig wasp species the sex ratio is negative related to the intensity ofLMC. When foundress number increases, the intensity of LMC de-creases while the sex ratio of fig wasps increases. In populationof high level inbreeding, the sex ratio is always female-biased [6,7].

More than 800 species of figs exist on this planet. A single figspecies has one to four pollinator species and also hosts up to 30non-pollinating wasp species [8]. High densities of non-pollinatingwasps could distort the sex ratio of pollinating species if non-poll-inators cause the mortality of one sex of the pollinators [9]. NPFWsare very important factors that affect fig wasps sex ratio, but al-ways ignored. Peng studied Ficus hispida, and found NPFWs directlyreduced the number of pollinator, but did not changed their sex ra-tio obviously [10]; Pereira studied Ficus ccitrifolia in brazil andfound NPFWs distorted pollinator’s sex ratio directly. However hedid not provide an explanation [9]. In this study, we focus onhow NPFWs distort pollinator fig wasps’ original sex ratio, andtry to explain the mechanism by showing the spatial distributionpatterns of three fig wasps.

2. Research material and methods

2.1. Research material

We used more than 120 syconia the fig Ficus semicordata inXishuangbanna.

F. semicordata are pollinated by host-specific wasps Ceratosolengravely (Agaonidae). A total of 4 NPFWs exist on F. semicordata, thatare Philotrypesis dunta, Platyneura cunia, Sycoscapter trifemmensisand Apocrypta sp. They all parasite on C. gravely.

2.2. Research methods

2.2.1. Investigation of ovipositing behavior and sequenceSample plot is located in XTBG which lies in tropical area of

Xishuangbanna. We observed the oviposition timing of all figwasps exist on F. semicordata from September to December 2009.

2.2.2. Controlling experiment of pollinator fig waspInsulate figs by yarn bags (20 � 25 cm) in order to prevent fig

wasps’ ovipositon. When figs get into acceptance stage, we startintroducing pollinator fig wasp into figs: Firstly, we collect maturemale figs and keep them in yarn bags to wait fig wasps get out ofgalls. Usually, fig wasps come out in the morning of next day. Thenwe quickly take them to sample figs and begin to introduce them

into right figs. After that, we insulate the figs by yarn bags again.Our experiment has 3 treatments, 1 foundress, 2 foundress and 3foundress respectively. Each treatment has at least 20 figs. All sam-ple syconia were collected early in the male flower phase to ensurethat all offspring wasps had not yet to emerge from their galls.Immediately after collection, all syconia were placed in 75% etha-nol. Long style flowers always has a short pedicel, while short styleflowers always has a long pedicel [7,11,12]. In our experiment, wesplit a fig into four pieces and choose one piece randomly. Pediceland ovary length are measured before we take fig wasps out of theirgalls to check the sex.

2.2.3. Controlling experiment of non-pollinator S. trifemmensisInsulate figs by yarn bags in order to prevent fig wasps’ oviposi-

ton as we do in the previous experiment. When figs get into accep-tance stage, we start introducing fig wasps into figs: First, weintroduce two pollinators into each figs and wait for the stageS. trifemmensis comes. When S. trifemmensis come forth, we startintroduce 1, 5 and 10 fig wasps into different figs. Those 3 differenttreatments each has at least 20 figs. When figs nearly get into mat-uration while fig wasps still in their galls, we collect figs and putthem into 75% ethanol. In our experiment, we split a fig into fourpieces and choose one piece randomly. Pedicel length is measuredbefore we take fig wasps out of galls to check the species and the sex.

2.2.4. Controlling experiment of NPFWsInsulate figs by yarn bags in order to prevent fig wasps’ ovipos-

iton as we do in the previous experiment. When figs get into accep-tance stage, we start introducing fig wasps into figs: Fist, weintroduce two pollinators into each figs and wait for differentNPFWs to come. We introduce Apocrypta sp. P. cunia andS. trifemmensis into different figs successively. Those 3 differenttreatments each has at least 20 figs Pedicel length is measured be-fore we take fig wasps out of galls to check the species and the sex.

2.3. Data analyse

Data are recorded in Excel 2003 before using SPSS (13.0) toanalyze. Charts are made by Sigma Plot (10.0) or SPSS (13.0).

3. Results

3.1. Ovipositing sequence of all fig wasps

Oviposition timing of all fig wasps exist on F. semicordata areinvestigated and we found there are bit of differences comparedto previous studies. Different species of fig wasps turn up on differ-ent stages during fig’s growth. Pollinator is the firth to come, andaccording to their population size, each fig has 0–12 pollinators.P. dunta turns out on the third day after pollinators come to figsP. dunta is hardly to be noticed because of their small population,and they always show up only 1 day. Apocrypta sp. is the secondNPFWs to come, and them also has a small population. Howeverthey can oviposit for about 4 days. P. cunia is the forth fig waspscome to oviposit on the syconia, and that is 10 days after pollina-tors. Their population size is the second largest. The last one toemerge is S. trifemmensis. S. trifemmensis oviposits much earlierthan we used thought, 14–32 days later than the pollinators. Theaverage ovipositor length of four NPFWs increases along with theirappearance order.

3.2. Relationship between foundress number and their offspring’sspacial distribution

Results shows offspring number in each fig does not increaselinear with foundress number from 1 to 3. Offspring number comes

Fig. 1. Relationship between total number of offspring (mean ± SE) and averagenumber of offspring in different foundress broods.

Fig. 2. Spacial distribution of offspring in different foundress.

Fig. 3. Relationship between spacial distribution of S. trifemmensis and foundressnumber.

X. Yan et al. / Acta Ecologica Sinica 32 (2012) 99–103 101

to a peak when foundress number reaches 2, that is (279.4 ± 22.84)(n = 10) and so different from other two treatments (F2, 24 = 3.567,P = 0.044). Offspring number gets to (209.8 ± 18.62) and (206.1 ±26.33) when foundress number reaches 1 (n = 10) and (n = 7), butthat does not makes any differences. The reason of this instancemay be the competing behavior such as fighting for the reproduc-tion chances deplete foundress’ energy or disturb others’ oviposit-ing. All the same time, the average offspring number of eachfoundress decreases sharply when foundress number increases to3 (F2, 24 = 21.534, P < 0.01) (Fig. 1).

Then the spatial location of male and female progeny of the pol-linator was examined (Fig. 2). We found foundress prefer to use in-ner ovules. Only when offspring number starts to increase, theouter ovules can be used. When foundress number is 1, offspringnumber is the lowest, and T shows pedicel length has no distinctdifference between sexes (t = 1.89, P = 0.087). When foundressnumber reaches 3, there is distinct difference between male and fe-male pollinator fig wasps (t = 6.027, P < 0.001). Nevertheless, off-spring number come to a peak when foundress number arrives at2, and the pedicel length of different sex of fig wasps is notable dif-ferent from each other (t = 7.431, P < 0.001).

The pedicel length of galls fig wasps stay in minishes when off-spring number gets increase. But the galls male fig wasps stay inchanges little (F2, 655 = 11.368, P < 0.001), while the female’schanges more (F2, 2534 = 127.115, P < 0.001). Consequently, moreand more female pollinator offspring were spread to other parts ofa syconia, while female offspring were more developed near the figcavity. The more pollinator offspring produced, the more clear thisphenomenon wound be.

Fig. 4. Different spacial distribution of two non-pollinator on different ovipostiondate.

3.3. Spacial distribution of two NPFWs

Spacial distribution of S. trifemmensis has no clear relationshipwith foundress number. More ovaries with long pedicel areexploited when foundress number increases, but their spacialdistribution varies little (Fig. 3). There is no remarkable differencewhen compare the average pedicel length under the condition of 1foundress, 2 foundress and 3 foundress (F2, 342 = 2.027, P = 0.133),thought the distribution range broadened. These experimentsdemonstrate that NPFWs are restricted by their ovipositor length,and they prefer to, or in other words, can only lay their eggs intoovules near the fig wall.

We surveyed the spacial distribution of two NPFWs (S. trifemm-ensis and P. cunia) that we introduced onto figs at different timepoints, and found the variation tendency of spacial distributionare totally contrary (Fig. 4). The average pedicel length of flowers

occupied by S. trifemmensis gets slightly shorter when we put offthe introducing time point. (F2, 195 = 0.44, P = ns). This meansmore fig wasps are distributed near the fig wall. The cause lies inthe fact that fig wasps can not reach to ovaries with long pedicelas flowers keep growing all the time. Meanwhile, the average ped-icel length of flowers occupied by P. cunia gets slightly longer whenwe put off the introducing time point. (F2, 195 = 0.44, P = ns). Thismeans more fig wasps are distributed near the fig caving.

Fig. 5. Relative spacial distribution of different sex of pollinater and non-pollinater.

102 X. Yan et al. / Acta Ecologica Sinica 32 (2012) 99–103

3.4. Spacial distribution contrast between NPFWs and pollinator figwasp

Comparing spacial distribution between NPFWs and pollinatorfig wasp (Fig. 5). We found NPFWs are more concentrated to shortpedicel flowers (1.83 ± 0.78 mm), male pollinator fig wasps assem-ble to long pedicel flowers (2.79 ± 1.03 mm), and the last femalepollinator fig wasps gather in the middle (2.31 ± 0.98 mm). ANOVAshows there is notable differences between these 3 treatment(F2, 1729 = 84.488, P < 0.001). This pattern of segregation of male lar-vae gall in inner ovules and female larvae gall in outer ovules sug-gests that female offspring are vulnerable to attack by parasiticwasps that oviposit from outside the syconium. This means theresulting gradient in offspring viability between male and femalecontributes to selection on pollinators’ less female-biased sexratio. Partial correlation analyse shows that sex ratio of pollina-tor’ progeny has a positive relationship with the number ofNPFWs(r = 0.538�, P = 0.018). Exclude the affection of NPFWs, thesex ratio of pollinator was not in agreement with local matecompetition theory perfectly, but it was still female-biased.

4. Discussions

First, we observed the oviposition timing of all fig wasps existon F. semicordata and found there are bit of differences comparedto previous studies [13,14]. In previous studies, P. cunia and Apo-crypta sp. is the third and forth fig wasps come to figs to oviposit.Moreover, S. trifemmensis come to oviposit lately during growingphase. But in my experiments, P. cunia is the forth fig wasps cometo oviposit on the syconia, and that is 10 days after pollinators.S. trifemmensis oviposits much earlier than we used thought,14–32 days later than the pollinators.

Then the spatial location of male and female progeny of the pol-linator was examined. We found foundress prefer to use innerovules. Only when offspring number starts to increase, the outerovules can be used. More male pollinator offspring were developednear the fig cavity, while female offspring were more spread toother parts of a syconia. The more pollinator offspring produced,the more clear this phenomenon wound be. This kind distributionpattern is related to fig wasps’ oviposition, which is related to fig’sphysical structure [15]. Fig syconia is rounded and hollow. A largenumber of flowers sprout inside from the fig wall. Long style flow-ers always has a short pedicel, while short style flowers always hasa long pedicel [11,12]. Ovules are the places where fig wasps laytheir eggs. Pollinators fig wasps prefer to use inner ovules. Theyavoid or are prevented from ovipositing into outer ovules [17]. Dif-ferent pedicel length causes ovaries of different distances to figwall. It is easier for NPFWs to use short pedicel flowers near fig

wall, while pollinator fig wasps use long pedicel flowers in the nearthe center of the syconium easily. Raja found foundress always laymale offspring before lay female offspring in the experiment of poi-soning foundress at different time points. This phenomenon isusual, and the reason is still unknown [16]. But the tactics to laymale offspring first may cause they all laid in ovaries with shortpedicel [5]. This kind oviposition mode makes male and femalepollinator distributed in different layers.

Based on this distribution pattern, NPFWs has a major influenceon pollinator’s sex ratio. Cause parasitism risk decreases towardsthe center of the syconium, where inner ovules provide enemy-freespace for most of male pollinator offspring. We suggest that theresulting gradient in offspring viability between male and femalecontributes to selection on pollinators’ less female-biased sex ratio.Moreover, Dunn found it is easier for pollinator to use flowers nearthe center of the syconium [17]. This pattern of segregation of malelarvae gall in inner ovules and female larvae gall in outer ovulessuggests that female offspring are vulnerable to attack by parasiticwasps that oviposit from outside the syconium. Experiments hasdemonstrate that NPFWs prefer to lay their eggs into ovules nearthe fig wall [18]. In this way, the competition between pollinatorand NPFWs focus on ovules near the fig wall.

In this study, the distribution of S. trifemmensis varies littlewhen foundress number increases. The cause of this instancemay lies in these two reasons. One is that NPFWs are not restrictedby their ovipositor length, and they just happened to lay their eggsinto female pollinators; the other is that our NPFWs sample is notenough abundant comparing to the population of pollinator. It isunable to show us the truth. But when we surveyed the spacial dis-tribution of S. trifemmensis and P. cunia that we introduced ontofigs at different time points, we found the variation tendency ofspacial distribution are totally contrary (Fig. 4). The reason maybe the ovipositing endurance time differ widely between thosetwo NPFWs. Ovipositiong time of S. trifemmensis can lasts 12 days,and during this long time pedicel length keep growing to a extentthat prevent fig wasps’ deep exploiture; P. cunia can oviposit onlyfor 2 days, and in such a short time figs can not grow much bigger.In addition to this, sample size of P. cunia is really few. So, the truthis not fully revealed. In all probability, NPFWs are restricted bytheir ovipositor length, and they can only lay their eggs into ovulesnear the fig wall.

Finally, we examined the spatial location of NPFWs and com-pared it with male/female progeny of pollinator. What we foundwas that these three kind fig wasps are layered distributed, andNPFWs has a high possibility to parasitize in female pollinatorlarvae. Therefore, NPFWs have a great effect on the sex ratio ofthe pollinator. We suggest that the resulting gradient in offspringviability between male and female contributes to selection onpollinators’ less female-biased sex ratio.

Acknowledgements

This research was funded by the Chinese Natural Science Foun-dation (30970439, 30970403, 31120002) and Candidates of theYoung and Middle Aged Academic Leaders of Yunnan Province(2011HB041).

References

[1] S. Raja, N. Suleman, S.G. Compton, J.C. Moore, The mechanism of sex ratioadjustment in a pollinating fig wasp, Proceedings of the Royal Society B:Biological Sciences 275 (1643) (2008) 1603–1610.

[2] S.A. Frank, Hierarchical selection theory and sex-ratios II. On applying thetheory, and a test with fig wasps, Evolution 39 (5) (1985) 949–964.

[3] E.A. Herre, Optimality, plasticity and selective regime in fig wasp sex-ratios,Nature 329 (6140) (1987) 627–629.

[4] S.A. West, M.G. Murray, C.A. Machado, A.S. Griffin, E.A. Herre, Testing Hamilton’srule with competition between relatives, Nature 409 (6819) (2001) 510–513.

X. Yan et al. / Acta Ecologica Sinica 32 (2012) 99–103 103

[5] G.D. Weiblen, How to be a fig wasp, Annual Review Entomology 47 (2002)299–330.

[6] E.A. Herre, Sex-ratio adjustment in fig wasps, Science 228 (4701) (1985) 896–898.

[7] F. Kjellberg, J.L. Bronstein, G. van Ginkel, J.M. Greeff, J.C. Moore, N. Bossu-Dupriez, M. Chevolot, G. Michaloud, Clutch size: a major sex ratio determinantin fig pollinating wasps?, Comptes Rendus Biologies 328 (5) (2005) 471–476.

[8] J.M. Cook, S.T. Segar, Speciation in fig wasps, Ecological Entomology 35 (2010)54–66.

[9] R.A.S. Pereira, A.P. Do Prado, Non-pollinating wasps distort the sex ratio ofpollinating fig wasps, Oikos 110 (3) (2005) 613–619.

[10] Y.Q. Peng, D.R. Yang, Q.Y. Wang, Adjustment and stabilization of sex ratioin Ceratosolen solmsi marchali, Acta Ecologica Sinica 25 (6) (2005)1347–1351.

[11] R.J.C. Nefdt, S.G. Compton, Regulation of seed and pollinator production in thefig fig wasp mutualism, Journal of Animal Ecology 65 (2) (1996) 170–182.

[12] M.C. Anstett, J.L. Bronstein, M. Hossaert-McKey, Resource allocation: a conflictin the fig/fig wasp mutualism?, Journal of Evolutionary Biology 9 (4) (1996)417–428

[13] Q.Y. Wang, D.R. Yang, Y.Q. Peng, Pollination behaviour and propagation ofpollinator wasps on Ficus semicordata in Xishuangbanna, China, ActaEntomologica Sinica 47 (1) (2003) 27–34.

[14] W.Q. Zhen, D.W. Huang, D.R. Yang, C.D. Zhu, Fig wasps associated with Ficussemicordata: oviposition timing and their population relationship, ActaEntomologica Sinica 47 (6) (2004) 787–792.

[15] M. Zavodna, S.G. Compton, S. Raja, P.M. Gilmartin, J.M.M. van Damme, Do figwasps produce mixed paternity clutches?, Journal of Insect Behavior 18 (3)(2005) 351–362

[16] M. Kinoshita, E. Kasuya, T. Yahara, Effects of time-dependent competition foroviposition sites on clutch sizes and offspring sex ratios in a fig wasp, Oikos 96(1) (2002) 31–35.

[17] D.W. Dunn, S.T. Segar, J. Ridley, R. Chan, R.H. Crozier, D.W. Yu, J.M. Cook, A rolefor parasites in stabilising the fig-pollinator mutualism, PLoS Biology 6 (3)(2008) 490–496.

[18] Z.J. Wang, F.P. Zhang, Y.Q. Peng, L.F. Bai, D.R. Yang, Comparison of reproductivestrategies in two externally ovipositing non-pollinating fig wasps, Symbiosis51 (2) (2010) 181–186.