Seed dormancy and germination of Ficus lundellii and tropical forest restoration

We investigated seed dormancy and germination in Ficus lundellii Standl. (Moraceae), a native species of Mexico's Los Tuxtlas tropical rain forest. In an 8-h photoperiod at an alternating diurnal (16/8 h) temperature of 20/30 degrees C, germination was essentially complete (96%) within 28 days, whereas in darkness, all seeds remained dormant. Neither potassium nitrate (0.05-0.2%) applied continuously nor gibberellic acid applied either continuously (10-200 ppm) or as a 24 hour pretreatment (2000 ppm) induced germination in the dark. Germination in the light was not reduced by a 24-h hydrochloric acid (0.1-1%) pretreatment, but it was reduced both by a 24-h pretreatment with either H2O2 (0. 1-5 M) or 5% HCl, or by more than 5 days of storage at 40 degrees C (4.5% seed water content). In a study with a 2-dimensional temperature gradient plate, seeds germinated fully and rapidly in the light at a constant temperature of 30 degrees C, and fully but less rapidly in the light at alternating temperatures with low amplitudes (< 12 degrees C) about the optimal constant temperature. The base, optimal and ceiling temperatures for rate of germination were estimated as 13.8, 30.1 and 41.1 degrees C, respectively. In all temperature regimes, light was essential for the germination of F lundellii seeds.

Phylogeny, biogeography, and ecology of Ficus section Malvanthera (Moraceae)

We conducted the first molecular phylogenetic study of Ficus section Malvanthera (Moraceae; subgenus Urostigma) based on 32 Malvanthera accessions and seven outgroups representing other sections of Ficus subgenus Urostigma. We used DNA sequences from the nuclear ribosomal internal and external transcribed spacers (ITS and ETS), and the glyceraldehyde-3-phosphate dehydrogenase (G3pdh) region. Phylogenetic analysis using maximum parsimony, maximum likelihood and Bayesian methods recovered a monophyletic section Malvanthera to the exclusion of the rubber fig, Ficus elastica. The results of the phylogenetic analyses do not conform to any previously proposed taxonomic subdivision of the section and characters used for previous classification are homoplasious. Geographic distribution, however, is highly conserved and Melanesian Malvanthera are monophyletic. A new subdivision of section Malvanthera reflecting phylogenetic relationships is presented. Section Malvanthera likely diversified during a period of isolation in Australia and subsequently colonized New Guinea. Two Australian series are consistent with a pattern of dispersal out of rainforest habitat into drier habitats accompanied by a reduction in plant height during the transition from hemi-epiphytic trees to lithophytic trees and shrubs. In contradiction with a previous study of Pleistodontes phylogeny suggesting multiple changes in pollination behaviour, reconstruction of changes in pollination behaviour on Malvanthera, suggests only one or a few gains of active pollination within the section. (C) 2008 Elsevier Inc. All rights reserved.

Microsatellite primers for Ficus racemosa and Ficus rubiginosa

We developed 11 polymorphic microsatellite loci each for the figs Ficus (Sycomorus) racemosa and Ficus (Urostigma) rubiginosa from AG- and TG-enriched genomic libraries. These 22 loci were investigated for cross-species amplification and polymorphism in 17–21 F. racemosa and 16–24 F. rubiginosa individuals from Townsville, Australia. Observed heterozygosities range from 0.12 to 0.90 in F. racemosa and from 0.25 to 1.0 in F. rubiginosa.

Intra- and interspecific variations in vacuolar flavonoids among Ficus species from the Budongo Forest, Uganda

Leaves of 14 species of Ficus growing in the Budongo Forest, Uganda, were analysed for vacuolar flavonoids. Three to six accessions were studied for each species to see whether there was intraspecific chemical variation. Thirty-nine phenolic compounds were identified or characterised, including 14 flavonol O-glycosides, six flavone O-glycosides and 15 flavone C-glycosides. In some species the flavonoid glycosides were acylated. Ficus thonningii contained in addition four stilbenes including glycosides. Most of the species could be distinguished from each other on the basis of their flavonoid profiles, apart from Ficus sansibarica and Ficus saussureana, which showed a very strong intraspecific variation. However, on the whole flavonoid profiles were sufficiently distinct to help in future identifications.

Chaos of Wolbachia Sequences Inside the Compact Fig Syconia of Ficus benjamina (Ficus: Moraceae)

Figs and fig wasps form a peculiar closed community in which the Ficus tree provides a compact syconium (inflorescence) habitat for the lives of a complex assemblage of Chalcidoid insects. These diverse fig wasp species have intimate ecological relationships within the closed world of the fig syconia. Previous surveys of Wolbachia, maternally inherited endosymbiotic bacteria that infect vast numbers of arthropod hosts, showed that fig wasps have some of the highest known incidences of Wolbachia amongst all insects. We ask whether the evolutionary patterns of Wolbachia sequences in this closed syconium community are different from those in the outside world. In the present study, we sampled all 17 fig wasp species living on Ficus benjamina, covering 4 families, 6 subfamilies, and 8 genera of wasps. We made a thorough survey of Wolbachia infection patterns and studied evolutionary patterns in wsp (Wolbachia Surface Protein) sequences. We find evidence for high infection incidences, frequent recombination between Wolbachia strains, and considerable horizontal transfer, suggesting rapid evolution of Wolbachia sequences within the syconium community. Though the fig wasps have relatively limited contact with outside world, Wolbachia may be introduced to the syconium community via horizontal transmission by fig wasps species that have winged males and visit the syconia earlier.

The effect of wilting and soaking Eupatorium adenophorum on its digestibility in vitro and voluntary intake by goats

An experiment was conducted to determine what effect simple treatments might have on the voluntary intake by goats in Nepal of Eupatorium adenophorum, an invasive weed that is usually only consumed by goats to a very limited extent. Samples of E. adenophorum were collected and either untreated, soaked for 2 h or wilted for 2 h before being oven dried (60 degrees C) and ground. Soaking and wilting had little effect on the chemical composition of E. adenophorum, but did increase (P=0.036) its in vitro organic matter degradability, by approximately 8%. The short-term intake rate (STIR) of treated and untreated E. adenophorum was then estimated with eight goats. Soaking time (from 2 to 24 h) was not related to STIR (r = -0.111, P=0.198), but the time E. adenophorum was left to wilt (from 2 to 48h), was positively related to STIR (r=0.521, P<0.001), with values of STIR (g dry matter/min kg goat liveweight(0.75)) being 0.405, 0.649,1.058, S.E.M. 0.088 for E. adenophorum, that had been wilted for 0, 24 and 48 h respectively (P<0.001). Liveweight change of goats and voluntary intake of E. adenophorum by goats was then estimated with 24 goats. E. adenophorum was fed either unwilted, or wilted for 24 or 48 h. It was fed as the sole forage or as a 3:1 mixture (dry matter basis) with Ficus cunia. There was a linear (P<0.001) and quadratic (P<0.01) increase in the intake of total forage and E. adenophorum with wilting time of E. adenophorum. Offering Ficus cunia increased total forage intake, but decreased E. adenophorum intake (P<0.05). After four weeks, there was virtually no change in goat liveweight and no significant difference between treatments. The results suggest that wilting E adenophorum for 24 h could increase its intake by goats, and thereby increase its usefulness, as a potential source of forage in the dry season of Nepal, when forage scarcity is a common constraint to livestock production. (C) 2007 Elsevier B.V. All rights reserved.

Characterization of microsatellite markers for Sycoscapter nonpollinating fig wasps

We isolated 18 microsatellites from Sycoscapter australis, a nonpollinating fig wasp that develops in figs of Ficus macrophylla, and assessed their variability in 20 wasps. We further optimized nine of these loci for use in three other Sycoscapter species that develop in Ficus rubiginosa figs and assessed their variability in 47-140 wasps per species. These are the first microsatellites developed for nonpollinating fig wasps and show sufficient polymorphism to become important tools in evolutionary and genetical studies of Sycoscapter wasps.

Male morphology and dishonest signalling in a fig wasp

Despite theoretical predictions, dishonest signalling has rarely been observed in aggressive interactions. We present evidence of such signalling in the nonpollinating. g wasp Philotrypesis sp. A ex Ficus rubiginosa. First, morphometric data indicated that an alternative 'atypical' male morph (17.8% of individuals) exists that tends to be larger in body size and has longer mandibles for a given body size than other 'typical' males. Second, behavioural observations suggested that males use mandible gape width (which depends on mandible length) as a cue to assess opponents before fights and retreat without escalating if they are unlikely to win, and, probably because their greater mandible gape width causes more opponents to retreat without escalating, that atypical males engaged in fewer fights than typical males for a given body size but had higher mating success. Third, atypical males were less likely to win fights than typical males of similar mandible length relative to opponents. In addition, we found that atypical males incur more injuries (greater receiver-dependent signal costs) than typical males of similar body size relative to rivals. We discuss the implications of our findings for future work on dishonest signalling. (C) 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Interference competition and high temperature reduce the ‘virulence’ of fig wasps and contribute to stability of a fig-wasp mutualism

Fig trees are pollinated by fig wasps, which also oviposit in female flowers. The wasp larvae gall and eat developing seeds. Although fig trees benefit from allowing wasps to oviposit, because the wasp offspring disperse pollen, figs must prevent wasps from ovipositing in all flowers, or seed production would cease, and the mutualism would go extinct. In Ficus racemosa, we find that syconia (‘figs’) that have few foundresses (ovipositing wasps) are underexploited in the summer (few seeds, few galls, many empty ovules) and are overexploited in the winter (few seeds, many galls, few empty ovules). Conversely, syconia with many foundresses produce intermediate numbers of galls and seeds, regardless of season. We use experiments to explain these patterns, and thus, to explain how this mutualism is maintained. In the hot summer, wasps suffer short lifespans and therefore fail to oviposit in many flowers. In contrast, cooler temperatures in the winter permit longer wasp lifespans, which in turn allows most flowers to be exploited by the wasps. However, even in winter, only in syconia that happen to have few foundresses are most flowers turned into galls. In syconia with higher numbers of foundresses, interference competition reduces foundress lifespans, which reduces the proportion of flowers that are galled. We further show that syconia encourage the entry of multiple foundresses by delaying ostiole closure. Taken together, these factors allow fig trees to reduce galling in the wasp-benign winter and boost galling (and pollination) in the wasp-stressing summer. Interference competition has been shown to reduce virulence in pathogenic bacteria. Our results show that interference also maintains cooperation in a classic, cooperative symbiosis, thus linking theories of virulence and mutualism. More generally, our results reveal how frequency-dependent population regulation can occur in the fig-wasp mutualism, and how a host species can ‘set the rules of the game’ to ensure mutualistic behavior in its symbionts.

Longevity, early emergence and body size in a pollinating fig wasp - implications for stability in a fig-pollinator mutualism

1. Fig trees (Ficus) are pollinated only by agaonid wasps, whose larvae also gall fig ovules. Each ovule develops into either a seed (when pollinated) or a wasp (when an egg is also laid inside) but not both. 2. Ovipositing wasps (foundresses) favour ovules near the centre of the enclosed inflorescence (syconium or 'fig'), leaving ovules near the outer wall to develop into seeds. This spatial stratification of wasps and seeds ensures reproduction in both partners, and thereby enables mutualism persistence. However, the mechanism(s) responsible remain(s) unknown. 3. Theory shows that foundresses will search for increasingly rare inner ovules and ignore outer ovules, as long as ovipositing in outer ovules is sufficiently slow and/or if inner ovules confer greater fitness to wasps. The fig-pollinator mutualism can therefore be stabilized by strong time constraints on foundresses and by offspring fitness gradients over variation in ovule position. 4. Female fig wasps cannot leave their galls without male assistance. We found that females in outer ovules were unlikely to be released. Inner ovules thus have added value to foundresses, because their female offspring are more likely to mate and disperse. 5. For those offspring that did emerge, gall position (inner/outer) and body size did not influence the order in which female pollinators exited syconia, nor did early emerging wasps enjoy increased life spans. 6. We also found that the life spans of female wasps nearly doubled when given access to moisture. We suggest that conflict resolution in the fig-pollinator mutualism may thus be influenced by tropical seasonality, because wasps may be less able to over-exploit ovules in dry periods due to time constraints.

A role for parasites in stabilising the fig-pollinator mutualism

Mutualisms are interspecific interactions in which both players benefit. Explaining their maintenance is problematic, because cheaters should outcompete cooperative conspecifics, leading to mutualism instability. Monoecious figs (Ficus) are pollinated by host-specific wasps (Agaonidae), whose larvae gall ovules in their "fruits'' (syconia). Female pollinating wasps oviposit directly into Ficus ovules from inside the receptive syconium. Across Ficus species, there is a widely documented segregation of pollinator galls in inner ovules and seeds in outer ovules. This pattern suggests that wasps avoid, or are prevented from ovipositing into, outer ovules, and this results in mutualism stability. However, the mechanisms preventing wasps from exploiting outer ovules remain unknown. We report that in Ficus rubiginosa, offspring in outer ovules are vulnerable to attack by parasitic wasps that oviposit from outside the syconium. Parasitism risk decreases towards the centre of the syconium, where inner ovules provide enemy-free space for pollinator offspring. We suggest that the resulting gradient in offspring viability is likely to contribute to selection on pollinators to avoid outer ovules, and by forcing wasps to focus on a subset of ovules, reduces their galling rates. This previously unidentified mechanism may therefore contribute to mutualism persistence independent of additional factors that invoke plant defences against pollinator oviposition, or physiological constraints on pollinators that prevent oviposition in all available ovules.

The phenology and potential for self-pollination of two Australian monoecious fig species

In this preliminary study, the reproductive phenology of two monoecious fig species, Ficus racemosa and F. rubiginosa, was examined in tropical Australia. Syconia (inflorescences) occurred on both species all year round, but pre-floral and interfloral syconia were much commoner than the wasp-receptive and wasp-emitting phases in both species. The temporal overlap of the wasp-receptive and wasp-emitting phases on a single tree indicated that self-pollination was possible in both species and that pollinators may sometimes persist through multiple generations on one tree. This sexual phase overlap was commoner in F. rubiginosa than in F racemosa. The two species also differed in their general within-tree asynchrony, with a higher diversity of phases on F. rubiginosa than on F. racemosa. The time from syconium initiation to ripening was very similar in F. rubiginosa (mean = 48.51 days) and F. racemosa (mean = 43.53 days). However, there was much more variation within and between trees for F. rubiginosa. In addition, the wasp-receptive phase was found to last up to 5 days (rnean = 4.38) in F. rubiginosa. Such longevity should contribute substantially to local pollinator population persistence. Future work should use genetic studies to determine whether self-pollination is common in these fig species.

Fighting strategies in two species of fig wasp

Although theory exists concerning the types of strategies that should be used in contests over resources, empirical work explicitly testing its predictions is relatively rare. We investigated male fighting strategies in two nonpollinating. g wasp species associated with Ficus rubiginosa figs. In Sycoscapter sp. A, males did not assess each other before or during fights over mating opportunities. Instead,fights continued until the loser reached an energetic cost threshold that was positively correlated with its body size (fighting ability) and retreated. In Philotrypesis sp. B, pre fight assessment was indicated, with males attacking competitively inferior rivals to remove them from the competitor pool ( they then continued to do so until they reached a cost threshold that was again positively correlated with body size). Using data on species ecology, we discuss our findings with respect to theory on when different fighting strategies should evolve. We argue that the type of strategy used by a. g wasp species is determined by its relative benefits in terms of inclusive fitness. (c) 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Comparisons of host mitochondrial, nuclear and endosymbiont bacterial genes reveal cryptic fig wasp species and the effects of Wolbachia on host mtDNA evolution and diversity

Background Figs and fig-pollinating wasp species usually display a highly specific one-to-one association. However, more and more studies have revealed that the "one-to-one" rule has been broken. Co-pollinators have been reported, but we do not yet know how they evolve. They may evolve from insect speciation induced or facilitated by Wolbachia which can manipulate host reproduction and induce reproductive isolation. In addition, Wolbachia can affect host mitochondrial DNA evolution, because of the linkage between Wolbachia and associated mitochondrial haplotypes, and thus confound host phylogeny based on mtDNA. Previous research has shown that fig wasps have the highest incidence of Wolbachia infection in all insect taxa, and Wolbachia may have great influence on fig wasp biology. Therefore, we look forward to understanding the influence of Wolbachia on mitochondrial DNA evolution and speciation in fig wasps. Results We surveyed 76 pollinator wasp specimens from nine Ficus microcarpa trees each growing at a different location in Hainan and Fujian Provinces, China. We found that all wasps were morphologically identified as Eupristina verticillata, but diverged into three clades with 4.22-5.28% mtDNA divergence and 2.29-20.72% nuclear gene divergence. We also found very strong concordance between E. verticillata clades and Wolbachia infection status, and the predicted effects of Wolbachia on both mtDNA diversity and evolution by decreasing mitochondrial haplotypes. Conclusions Our study reveals that the pollinating wasp E. verticillata on F. microcarpa has diverged into three cryptic species, and Wolbachia may have a role in this divergence. The results also indicate that Wolbachia strains infecting E. verticillata have likely resulted in selective sweeps on host mitochondrial DNA.

Measuring the discrepancy between fecundity and lifetime reproductive success in a pollinating fig wasp

Lifetime reproductive success in female insects is often egg- or time-limited. For instance in pro-ovigenic species, when oviposition sites are abundant, females may quickly become devoid of eggs. Conversely, in the absence of suitable oviposition sites, females may die before laying all of their eggs. In pollinating fig wasps (Hymenoptera: Agaonidae), each species has an obligate mutualism with its host fig tree species [Ficus spp. (Moraceae)]. These pro-ovigenic wasps oviposit in individual ovaries within the inflorescences of monoecious Ficus (syconia, or ‘figs’), which contain many flowers. Each female flower can thus become a seed or be converted into a wasp gall. The mystery is that the wasps never oviposit in all fig ovaries, even when a fig contains enough wasp females with enough eggs to do so. The failure of all wasps to translate all of their eggs into offspring clearly contributes to mutualism persistence, but the underlying causal mechanisms are unclear. We found in an undescribed Brazilian Pegoscapus wasp population that the lifetime reproductive success of lone foundresses was relatively unaffected by constraints on oviposition. The number of offspring produced by lone foundresses experimentally introduced into receptive figs was generally lower than the numbers of eggs carried, despite the fact that the wasps were able to lay all or most of their eggs. Because we excluded any effects of intraspecific competitors and parasitic non-pollinating wasps, our data suggest that some pollinators produce few offspring because some of their eggs or larvae are unviable or are victims of plant defences.