Development and characterization of microsatellite markers for Liporrhopalum tentacularis Grandi,the pollinator fig wasp of Ficus montana Blume

Microsatellite markers for the pollinator fig wasp Liporrhopalum tentacularis were developed using genomic libraries enriched for di‐, tri‐ and tetranucleotide repeats. A subset of 31 positive clones was sequenced and primers were designed. Eleven primer pairs produced polymorphic amplification products in L. tentacularis. Eight markers gave unambiguously scorable patterns and were further characterized on 29 individuals collected from different fruits of the dioecious host fig Ficus montana in Indonesia. Three to 19 alleles per locus were detected in this set of samples. The observed heterozygosity ranged between 0.10 and 0.55.     

Parasitism of a pollinator fig wasp: mortalities are higher in figs with more pollinators,but are not related to local densities of figs

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A comparison of pollinator fig wasp development in figs of Ficus montana and its hybrids with Ficus asperifolia

Figs (Moraceae) and pollinator fig wasps (Hymenoptera: Agaonidae) have a highly specific mutualistic relationship but fig wasps occasionally enter atypical hosts, and this can lead to hybrid fig trees and the potential for gene flow between species. Many fig trees are dioecious, with fig wasp offspring developing in galled ovules inside figs on male trees, whereas seeds develop only in figs on female trees. We generated experimental hybrids between the Asian Ficus montana Blume and a closely related African species Ficus asperifolia Miquel. Male F1s were sterile if entered by Kradibia tentacularis (Grandi) (Agaonidae), the pollinator of F. montana, because its offspring always failed to develop, without ovule enlargement. As with the F1s, figs on most male backcross plants [F. montana × (F. montana × F. asperifolia)] also aborted shortly after pollinator entry, resulting in a higher turnover of figs than with F. montana, although the times taken for the figs to reach receptivity were similar. Pollinator larvae nonetheless consistently managed to develop inside the figs of one backcross plant and also occasionally in a few figs from another backcross individual. In these figs, galled ovules developed as normal, whereas in figs that aborted the galled ovules failed to enlarge. The sex ratio of K. tentacularis progeny in the backcross figs was female biased and did not differ from that in F. montana figs. Sycoscapter spec. (Hymenoptera: Pteromalidae), a parasitoid of K. tentacularis, was able to lay eggs and developed normally inside male backcross figs where its host was present.     

Dispersal of adult female fig wasps: 2. Movements between trees

Fig wasps (Chalcidoidea, Agaonidae, Agaoninae) are the exclusive pollinators of fig trees (Ficus spp., Moraceae). Fig development on the African fig tree, F. burtt-davyi, is normally synchronised on individual trees, but not between trees. Consequently the females of each generation of the pollinating species (Elisabethiella baijnathi) have to disperse to other trees to find ‘receptive’ figs which are suitable for oviposition. This paper examines this aspect of fig - fig wasp biology. The flight speed of insects is closely linked to their size, and directional flight is difficult for small insects, such as fig wasps, in all but the lightest of winds. We investigated the movements of fig wasps between trees using sticky traps placed around fig trees or near cotton bags containing figs. Away from the trees, the densities of flying wasps at different heights was also determined. When the wasps disperse from their natal figs they take off near-vertically. They are unable to exert directional control once they enter the air column and are subsequently blown downwind. Near receptive host trees the wasps appear to lose height and then fly upwind at speeds of around 25 cm/sec.     

Reconstruction of fig wasp mating structure: how many mothers share a fig?

Abstract.  1. Fig wasps (Hymenoptera: Agaonidae) represent an important model system for studies of sex ratio evolution, mainly because they may adjust their sex ratios in response to the numbers of ovipositing females (foundresses) that enter a fig and their clutch size.
2. Until recently, it was assumed that all foundresses fail to re-emerge from the figs that they have entered to oviposit, but there is increasing evidence that such re-emergence may be routine. The common practice of counting the number of dead foundresses present in a fig in order to deduce the number of foundresses is therefore questionable in species where failure to re-emerge has not been confirmed.
3. In this study, the alternative approach of microsatellite markers was used to reconstruct the within-fig breeding structure of a pollinating fig wasp by genetic analysis of the offspring. Broods of Liporrhopalum tentacularis , a species where foundresses regularly re-emerge from figs, were collected from figs of Ficus montana in their natural habitat in Indonesia as well as from an experimental glasshouse population in Leeds (U.K.).
4. The estimated foundress densities in the glasshouse population were similar to those in the field and ranged from one to six foundresses per brood.
5. Nearly 40% of all broods were produced by a single foundress, indicating that mating in these broods occurs exclusively between full siblings. High levels of inbreeding are therefore common in this species.     

Isolation of six microsatellite loci in the pollinating fig wasp,Platyscapa awekei

Platyscapa awekei DNA was enriched for several repeat motifs. Sequencing of 48 transformed colonies showed that 22 contained microsatellite loci. Eleven of these loci were tested and six of them proved to be reliable and variable. As expected from these wasps’ biology, F_IS was high (= 0.423), indicating frequent sibmating. Notwithstanding, we estimate that dispersing males may secure as much as 8% of all matings.     

Body size in a pollinating fig wasp and implications for stability in a fig‐pollinator mutualism

The fig–fig pollinator association is a classic case of an obligate mutualism. Fig‐pollinating wasps often have to fly long distances from their natal syconia to a receptive syconium and then must enter the narrow ostiole of the syconium to reproduce. Large wasps are expected to have a greater chance of reaching a receptive syconium. In this study, we tested this hypothesis and then examined whether the ostiole selectively prevented larger pollinators from entering the syconial cavity. In Xishuangbanna, China, Ceratosolen solmsi marchali Mayr (Hymenoptera: Agaonidae) pollinates the dioecious syconia of Ficus hispida L. (Moraceae). The body size of newly emerged wasps and wasps arriving at receptive syconia were compared. Wasps arriving at receptive syconia were significantly larger than newly emerged wasps. We also compared the size of wasps trapped in the ostiole with those in the cavity. Wasps trapped in the ostiole were significantly larger than those in the syconial cavity. Thus, in the case of F. hispida, large wasps were more likely to reach receptive syconia, but the ostiole limited maximum fig wasp size. This indicates that the ostiole, as a selective filter to pollinators, stabilizes pollinator size. Hence, it helps to maintain stability in the fig–fig pollinator mutualism.     

First record of an apparently rare fig wasp feeding strategy: obligate seed predation

1. Fig trees require host‐specific agaonid fig wasps for pollination, but their figs also support numerous non‐pollinating fig wasps (NPFW) that gall fig tissues or develop as parasitoids. 2. Ficus microcarpa L. is widely naturalised outside its native range and the most invasive fig tree species. Seed predators are widely used for the biological control of invasive plants, but no obligate seed predatory (as opposed to ovule or fig wall galling) NPFW have been recorded previously from any fig trees. 3. Philotrypesis NPFW are usually regarded as parasitoids or ‘inquilines’ (parasitoids that also eat plant material) of pollinator fig wasps, but the present study provides evidence that Philotrypesis taiwanensis, a NPFW associated with F. microcarpa, is an obligate seed predator: (i) adults emerge from seeds of typical appearance, with a surrounding elaiosome; (ii) it shows no preference for figs occupied by fig wasp species, other than the pollinator; (iii) it only develops in figs that contain pollinated ovules, avoiding figs occupied by an agaonid that fails to pollinate; (iv) larvae are distributed in layers where seeds are concentrated and (v) it has a negative impact on seed but not pollinator offspring numbers. 4. Philotrypesis is a large genus, and further species are likely to be seed predators.     

Development of 17 microsatellite markers for Ceratosolen constrictus, the pollinating fig wasp of Ficus fistulosa

The obligate mutualism between figs (Ficus) and fig pollinating wasps (Agaonidae) is regarded as a classic example of mutualism. Seventeen polymorphic microsatellite loci were developed for Ceratosolen constrictus, the pollinating wasp of the dioecious fig Ficus fistulosa. The number of alleles per locus ranged from two to 15 and the observed and expected heterozygosities ranged from 0.040 to 0.846 and from 0.040 to 0.916, respectively. These microsatellite loci offer a powerful tool for evolutionary and population genetic studies in C. constrictus, and gene flow of F. fistulosa.     

Maintenance of Specificity in an Isolated Fig

The obligate interaction between figs and their pollinating wasps is often cited as an extraordinary example of reciprocal species specificity and evolutionary cospeciation. However, recent studies have shown that breakdowns in one-to-one specificity are not rare (30–60% of species depending on the locality). Combined with evidence of hybridization in some species, this led researchers to propose that a better evolutionary model was one of groups of genetically well-defined pollinators coevolving with groups of frequently hybridizing figs. Nevertheless, these recent studies still indicate that a majority of fig species have one or more host-specific pollinator. The extent to which specificity barriers in these species are leaky will have important consequences for the evolutionary process in Ficus . At Lambir Hills N.P., Sarawak, a single individual of Ficus acamptophylla has become recently isolated from conspecifics through clearance of its specialized habitat, but adjacent forest has a diverse fig flora, including 16 species with congeneric pollinators. Thus, when this individual flowered I was able to investigate the maintenance of its specificity barriers in the absence of competition from the normal pollinator. Only 1 percent of inflorescences were entered by a single pollinator species, which had very low reproductive success, and no viable seeds were produced. Nonpollinating wasps also failed to reproduce in any of the inflorescences. These results indicate the maintenance of strict specificity barriers in this fig individual.     

Floral volatiles, pollinator sharing and diversification in the fig-wasp mutualism: insights from Ficus natalensis, and its two wasp pollinators (South Africa)

Combining biogeographic, ecological, morphological, molecular and chemical data, we document departure from strict specialization in the fig-pollinating wasp mutualism. We show that the pollinating wasps Elisabethiella stuckenbergi and Elisabethiella socotrensis form a species complex of five lineages in East and Southern Africa. Up to two morphologically distinct lineages were found to co-occur locally in the southern African region. Wasps belonging to a single lineage were frequently the main regional pollinators of several Ficus species. In South Africa, two sister lineages, E. stuckenbergi and E. socotrensis, pollinate Ficus natalensis but only E. stuckenbergi also regularly pollinates Ficus burkei. The two wasp species co-occur in individual trees of F. natalensis throughout KwaZulu-Natal. Floral volatile blends emitted by F. natalensis in KwaZulu-Natal were similar to those emitted by F. burkei and different from those produced by other African Ficus species. The fig odour similarity suggests evolutionary convergence to attract particular wasp species. The observed pattern may result from selection for pollinator sharing among Ficus species. Such a process, with one wasp species regionally pollinating several hosts, but several wasp species pollinating a given Ficus species across its geographical range could play an important role in the evolutionary dynamics of the Ficus-pollinating wasp association.     

The mechanism of pollinator specificity between two sympatric fig varieties: a combination of olfactory signals and contact cues

Background and Aims

Pollinator specificity facilitates reproductive isolation among plants, and mechanisms that generate specificity influence species boundaries. Long-range volatile attractants, in combination with morphological co-adaptations, are generally regarded as being responsible for maintaining extreme host specificity among the fig wasps that pollinate fig trees, but increasing evidence for breakdowns in specificity is accumulating. The basis of host specificity was examined among two host-specific Ceratosolen fig wasps that pollinate two sympatric varieties of Ficus semicordata, together with the consequences for the plants when pollinators entered the alternative host variety.

Methods

The compositions of floral scents from receptive figs of the two varieties and responses of their pollinators to these volatiles were compared. The behaviour of the wasps once on the surface of the figs was also recorded, together with the reproductive success of figs entered by the two Ceratosolen species.

Key Results

The receptive-phase floral scents of the two varieties had different chemical compositions, but only one Ceratosolen species displayed a preference between them in Y-tube trials. Specificity was reinforced at a later stage, once pollinators were walking on the figs, because both species preferred to enter figs of their normal hosts. Both pollinators could enter figs of both varieties and pollinate them, but figs with extra-varietal pollen were more likely to abort and contained fewer seeds. Hybrid seeds germinated at normal rates.

Conclusions

Contact cues on the surface of figs have been largely ignored in previous studies of fig wasp host preferences, but together with floral scents they maintain host specificity among the pollinators of sympatric F. semicordata varieties. When pollinators enter atypical hosts, post-zygotic factors reduce but do not prevent the production of hybrid offspring, suggesting there may be gene flow between these varieties.     

Putting your sons in the right place: the spatial distribution of fig wasp offspring inside figs

Abstract. 1. Pollinating fig wasps (Hymenoptera, Agaonidae) display sex ratio adjustment, producing less female‐biased combined sex ratios as the number of ovipositing females (foundresses) inside a fig increases. Because males have low mobility, the oviposition sites (galled ovules) chosen by each foundress are likely to have consequences for the mating structure of wasp populations within the figs. 2. In this study, the spatial location of male and female progeny of the pollinating fig wasp Liporrhopalum tentacularis developing within figs of its host plant Ficus montana was examined to investigate two questions: (i) are male and/or female wasp offspring clustered together or interspersed? and (ii) is their distribution affected by whether one or two foundresses are present? Microsatellite markers were used to identify the progeny of different foundresses in dual‐foundress figs. 3. More offspring developed in the central part of the figs, compared with the ostiolar and basal parts, irrespective of foundress number. Neither male nor female wasp offspring were clustered within a fig. 4. The sons of the second foundress to enter a fig were positioned at similar minimum distances to both sibling and non‐sibling females, whereas the sons of the first foundress were closer to their sibling females than to non‐sibling females. If male wasps mate predominantly with females in adjacent galls, then the positioning of sons by the second foundresses is beneficial for them both in terms of reduced sibling mating and because they are provided with ready access to the female progeny of the first foundress.     

Inbreeding and population structure in two pairs of cryptic fig wasp species

We used recently developed microsatellites to directly estimate inbreeding levels in two pairs of coexisting cryptic fig wasp species ('Pegoscapus hoffmeyeri sp. A and sp. B', 'P. gemellus sp. A and sp. B'). Previous tests of Hamilton's local mate competition (LMC) theory in fig wasps have used the number of dead foundresses in a fig fruit to indirectly estimate the relative contribution of each to the common brood and thereby the level of local mate competition. Further, the population level of inbreeding has been indirectly estimated using the distribution of foundress numbers across broods. Our direct genetic estimates confirmed previous assumptions that the species characterized by lower foundress numbers showed higher relative levels of inbreeding. However, there were quantitative differences between the observed level of inbreeding and the expectation based on the distribution of foundress numbers in both pollinator species associated with Ficus obtusifolia. Here, genotype compositions of broods revealed that only 23% of fruits with multiple foundresses actually contained brood from more than one foundress, thus explaining at least part of the underestimate of actual sibmating. Within the four wasp species there was no evidence for genetic differentiation among the wasp populations sampled from different trees across 20 km and from different points in time. Further, no genotypic disequilibrium was detected within any of the species. Although F1 hybrids were observed between the two species pollinating F. obtusifolia, there was no evidence of genetic introgression. Finally, we found that 11% of the sons of allospecifically mated mothers were diploid hybrids suggesting a break down of the sex determination system in hybrids.     

An inquiline fig wasp using seeds as a resource for small male production: a potential first step for the evolution of new feeding habits?

The processes allowing evolutionary transitions in resources used by parasitic wasps are largely unknown. Microhymenopteran communities associated with figs could provide a model system to investigate such transitions. We investigate here a species of genus Idarnes . The larvae generally develop as inquilines of the pollinating wasp larvae. However, in figs where the parasitic pressure is high, eggs are laid in developing seeds. These eggs turn into small males. This is the first report of seed consumption by a fig wasp. Using an alternative resource to produce small males could provide a pathway to select for increased ability to use this resource and hence provide an intermediate step for evolving the capacity to use new resources.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 9–17.     

The incidence and pattern of copollinator diversification in dioecious and monoecious figs

Differences in breeding system are associated with correlated ecological and morphological changes in plants. In Ficus, dioecy and monoecy are strongly associated with different suites of traits (tree height, population density, fruiting frequency, pollinator dispersal ecology). Although approximately 30% of fig species are pollinated by multiple species of fig‐pollinating wasps, it has been suggested that copollinators are rare in dioecious figs. Here, we test whether there is a connection between the fig breeding system and copollinator incidence and diversification by conducting a meta‐analysis of molecular data from pollinators of 119 fig species that includes new data from 15 Asian fig species. We find that the incidence of copollinators is not significantly different between monoecious and dioecious Ficus. Surprisingly, while all copollinators in dioecious figs are sister taxa, only 32.1% in monoecious figs are sister taxa. We present hypotheses to explain those patterns and discuss their consequences on the evolution of this mutualism.     

两种Eupristina属小蜂对宿主榕果挥发物和合成信息素的行为反应

高榕,雌雄同株,与其共生的形态高度相似的传粉小蜂Eupristina altissima与非传粉小蜂Eupristina sp.均为果内产卵。利用Y型嗅觉仪生物测定法研究E.altissima和Eupristina sp.对不同发育期高榕榕果挥发物以及合成信息素混合物的行为反应差异。结果表明:E.altissima和Eupristina sp.对高榕雌花期榕果挥发物具极显著趋向行为,对高榕雄花期榕果挥发物具极显著驱避行为,表明E.altissima与Eupristina sp.雌蜂能够敏锐辨别高榕雌花期和雄花期榕果的挥发物,快速远离雄花期榕果,并实现对宿主雌花期榕果的定位。但是,E.altissima与Eupristina sp.对其它发育时期榕果挥发物的行为反应具有差异性,前者对雌前期榕果和传粉或产卵后榕果释放的挥发物存在显著的趋避反应,而后者无明显的行为偏向性,表明E.altissima能够识别雌前期榕果和传粉或产卵后榕果,可避免误入其中而无法繁殖后代,Eupristina sp.则不具备这种识别能力。E.altissima和Eupristina sp.小蜂对合成信息素的行为反应也高度相似,表现为芳樟醇、苯乙烯,以及苯乙烯和金合欢醇的交互作用对两种小蜂选择行为影响显著,组合A_1B_2C_2(0.5%Linalool+1%Benzyl ethylene+1%Farnesol)的混合信息素对两种小蜂具有极显著的驱避作用,而组合A_2B_1C_1(1%Linalool+0.5%Benzyl ethylene+0.5%Farnesol),A_2B_1C_2(1%Linalool+0.5%Benzyl ethylene+1%Farnesol),A2B2C1(1%Linalool+1%Benzyl ethylene+0.5%Farnesol)的混合信息素对其具有极显著的吸引作用,但两种小蜂对组合A_1B_1C_1(0.5%Linalool+0.5%Benzyl ethylene+0.5%Farnesol)的行为反应存在差异。两种小蜂对榕果挥发物和合成信息素的反应异同,可能与其触角结构的差异密切相关。研究结果为探究榕-蜂共生体系的化学信号传导机制提供科学依据。     

Reproductive strategies of two forms of flightless males in a non‐pollinating fig wasp under partial local mate competition

1. The underlying basis of alternative male reproductive strategies is either genetic or environmental. Several non‐pollinating fig wasp lineages have dimorphic males, typically with winged males that disperse from natal figs to mate and flightless males that seek mating opportunities in natal figs. 2. Walkerella sp. from Ficus benjamina has dark and pale wingless males. Observations and experiments in Xishuangbanna, Southern China found that (i) the sex ratio of Walkerella sp. did not vary with foundress number or brood size. (ii) The frequency of dark males increased with brood size and foundress number and they were absent from figs with a single foundress. This produced a higher proportion of dark males at higher densities. (iii) Males of both morphs fought, but injuries to dark males were more frequent. (iv) Dark males were more likely to disperse away from their natal figs and (v) they were more resistant to dehydration. 3. Responses to selection are constrained by the genetic options available. Consequently, selection pressures acting on different lineages can produce similar outcomes that are achieved in different ways. Walkerella species lack winged males, but dark males display some of their features, dispersing from natal figs and displaying appropriate physiological and behavioural adaptations. However, dark males also displayed increased levels of damage from fighting – a feature unlikely to be shared with the winged males of other species.     

Predation on the pollinating fig wasp of Ficus erecta by larvae of Silba sp. (Diptera: Lonchaeidae)

This study examined the insect assemblage within the syconium of the dioecious fig Ficus erecta Thunb., 1786 (Moraceae) on Kii‐Oshima Island, Wakayama Prefecture, Japan. Species present included pollinating and non‐pollinating fig wasps Blastophaga nipponica Grandi, 1921 and Sycoscapter inubiae Ishii, 1934, respectively, as well as an undetermined species of Silba Macquart, 1851 (Diptera: Lonchaeidae). Larvae of Silba sp. were found only in fig syconia containing B. nipponica. Observations revealed that larvae of Silba sp. fed on female adults of B. nipponica within fig galls by thrusting their heads through the intersegmental membrane. Furthermore, larvae of Silba sp. had a high predation rate (90%). These observations suggest that larvae of Silba sp. are specialized for feeding on B. nipponica and have a negative effect on populations of this species.     

Diversification in the use of resources by Idarnes species: bypassing functional constraints in the fig–fig wasp interaction

Mutualisms such as the fig–fig wasp mutualism are generally exploited by parasites. We demonstrate that amongst nonpollinating fig wasps (NPFWs) parasitic on Ficus citrifolia, a species of Idarnes galls flowers and another species feeds on galls induced by other wasps killing their larvae. The galling wasp inserts its ovipositor through the fig wall into the fig cavity. The ovipositor then follows a sinuous path and is introduced through the stigma and style of the flower. The egg is deposited between the integument and nucellus, in the exact location where the pollinating mutualistic wasp would have laid its egg. Gall induction is a complex process. In contrast, the path followed by the ovipositor of the other species is straightforward: attacking a larva within a developed gall poses different constraints. Shifts in feeding regime have occurred repeatedly in NPFWs. Monitoring traits associated with such repeated evolutionary shifts may help understand underlying functional constraints. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 114–122.