Dipterocarpoidae genomics reveal their demography and adaptations to Asian rainforests.

Dipterocarpoideae species form the emergent layer of Asian rainforests. They are the indicator species for Asian rainforest distribution, but they are severely threatened. Here, to understand their adaptation and population decline, we assemble high-quality genomes of seven Dipterocarpoideae species including two autotetraploid species. We estimate the divergence time between Dipterocarpoideae and Malvaceae and within Dipterocarpoideae to be 108.2 (97.8‒118.2) and 88.4 (77.7‒102.9) million years ago, and we identify a whole genome duplication event preceding dipterocarp lineage diversification. We find several genes that showed a signature of selection, likely associated with the adaptation to Asian rainforests. By resequencing of two endangered species, we detect an expansion of effective population size after the last glacial period and a recent sharp decline coinciding with the history of local human activities. Our findings contribute to understanding the diversification and adaptation of dipterocarps and highlight anthropogenic disturbances as a major factor in their endangered status.

Olfactory and gustatory receptor genes in fig wasps: Evolutionary insights from comparative studies.

The mechanisms of chemoreception in fig wasps (Hymenoptera, Agaonidae) are of primary importance in their co-evolutionary relationship with the fig trees they pollinate. We used transcriptome sequences of 25 fig wasps in six genera that allowed a comparative approach to the evolution of key molecular components of fig wasp chemoreception: their odorant (OR) and gustatory (GR) receptor genes. In total, we identified 311 ORs and 47 GRs, with each species recording from 5 to 30 OR genes and 1-4 GR genes. 304 OR genes clustered into 18 orthologous groups known to be sensitive to cuticular hydrocarbons (CHC), pheromones, acids, alcohols and a variety of floral scents such as cineole, Linalool, and Heptanone. 45 GR genes clustered into 4 orthologous groups that contain sweet, bitter, CO2 and undocumented receptors. Gene sequences in most orthologous groups varied greatly among species, except for ORco (60.0% conserved) and sweet receptors (30.7% conserved). Strong purifying selection of both odorant and gustatory genes was detected, as shown by low ω values. Signatures of positive selection were detected in loci from both OR and GR orthologous groups. Fig wasps have relatively few olfactory and especially gustatory receptors, reflecting the natural history of the system. Amino acid sequences nonetheless vary significantly between species and are consistent with the phylogenetic relationships among fig wasps. The differences in ORs within some orthologous groups from the same species, but different hosts and from closely related species from one host can reach as low as 49.3% and 9.8% respectively, implying the ORs of fig wasps can evolve rapidly to novel ecological environments. Our results provide a starting point for understanding the molecular basis of the chemosensory systems of fig wasps.

Association of Fig Pollinating Wasps and Fig Nematodes inside Male and Female Figs of a Dioecious Fig Tree in Sumatra, Indonesia.

Nematodes can grow within the inflorescences of many fig trees (Ficus spp., Moraceae); however, the feeding behaviour of most nematodes is not known. Fig pollinating wasps (Hymenoptera: Agaonidae) transfer nematodes into young figs upon the wasps' entry into the figs to deposit their eggs. Most Asian fig trees, however, are functionally dioecious, and the pollinating wasps that enter female figs are unable to reproduce. They fail to produce the offspring required to carry the new generations of nematodes. We examined whether female figs of F. hispida can nonetheless support the development of phoretic nematode populations. Nematodes were extracted from male and female figs sampled in Sumatra, Indonesia, to compare the growth of their populations within the figs. We found three species of nematodes that grew within figs of male and female trees of F. hispida: Ficophagus cf. centerae (Aphelenchoididae), Martininema baculum (Aphelenchoididae) and Caenorhabditis sp (Rhabditidae). The latter species (Caenorhabditis sp.) has never been reported to be associated with F. hispida before. Nematode populations peaked at around 120-140 individuals in both sexes of figs, at the time when a succeeding generation of adult fig wasps appeared within male figs. The female figs could support the growth and reproduction of the three nematodes species; however, the absence of vectors meant that female figs remained as traps from which there could be no escape.

Asymmetric sharing of pollinator fig wasps between two sympatric dioecious fig trees: a reflection of supply and demand or differences in the size of their figs?

BACKGROUND: Host specificity among pollinator fig wasps (Agaonidae) depends on host plant specific volatile cues, but fig wasps must also pass through a narrow physical barrier (the ostiole) if they are to pollinate and oviposit. Across South East Asia the dioecious shrub Ficus hirta is associated with at least ten pollinator species allied to Valisia javana. Ficus triloba has a single recorded pollinator, Valisia esquirolianae. Receptive figs of F. hirta are usually much smaller than those of F. triloba, but at a mainland site where F. hirta has atypically large figs we identified both V. esquirolianae and V. javana from both Ficus species using COI and ITS2 sequencing. To investigate whether this host overlap was exceptional we reared fig wasps from the two trees elsewhere and recorded features that may facilitate host transfer between them, including attractant volatiles, reproductive phenology and the sizes of their figs and fig wasps. RESULTS: The two Ficus species were found to support both Valisia species at several of the sites, suggesting that the differences we detected in volatile profiles, ostiole sizes and pollinator head sizes are not strict barriers to host sharing. Valisia javana colonised F. triloba more frequently than V. esquirolianae colonised F. hirta. CONCLUSIONS: This asymmetric sharing of pollinators may reflect the relative abundance of the two species of fig wasps and differences in host reproductive phenology. Asynchronous flowering of individual F. hirta may favor local retention of pollinators, in contrast to the tree-wide synchrony of F. triloba figs, which can generate local shortages of V. esquirolianae. If the pollinator sharing by male figs of F. triloba and F. hirta also occurs in female figs then this could result in gene flow between them.

More examples of breakdown the 1:1 partner specificity between figs and fig wasps.

BACKGROUND: The obligate mutualism between fig trees (Ficus, Moraceae) and pollinating fig wasps (Agaonidae) is a model system for studying co-evolution due to its perceived extreme specificity, but recent studies have reported a number of examples of trees pollinated by more than one fig wasp or sharing pollinators with other trees. This will make the potential of pollen flow between species and hybridization more likely though only few fig hybrids in nature have been found. We reared pollinator fig wasps from figs of 13 Chinese fig tree species and established their identity using genetic methods in order to investigate the extent to which they were supporting more than one species of pollinator (co-pollinator). RESULTS: Our results showed (1) pollinator sharing was frequent among closely-related dioecious species (where pollinator offspring and seeds develop on different trees); (2) that where two pollinator species were developing in figs of one host species there was usually one fig wasp with prominent rate than the other. An exception was F. triloba, where its two pollinators were equally abundant; (3) the extent of co-pollinator within one fig species is related to the dispersal ability of them which is stronger in dioecious figs, especially in small species. CONCLUSIONS: Our results gave more examples to the breakdown of extreme specificity, which suggest that host expansion events where pollinators reproduce in figs other than those of their usual hosts are not uncommon among fig wasps associated with dioecious hosts. Because closely related trees typically have closely related pollinators that have a very similar appearance, the extent of pollinator-sharing has probably been underestimated. Any pollinators that enter female figs carrying heterospecific pollen could potentially generate hybrid seed, and the extent of hybridization and its significance may also have been underestimated.

Making the most of your pollinators: An epiphytic fig tree encourages its pollinators to roam between figs.

Ficus species are characterized by their unusual enclosed inflorescences (figs) and their relationship with obligate pollinator fig wasps (Agaonidae). Fig trees have a variety of growth forms, but true epiphytes are rare, and one example is Ficus deltoidea of Southeast Asia. Presumably as an adaptation to epiphytism, inflorescence design in this species is exceptional, with very few flowers in female (seed-producing) figs and unusually large seeds. Figs on male (pollinator offspring-generating) trees have many more flowers. Many fig wasps pollinate one fig each, but because of the low number of flowers per fig, efficient utilization by F. deltoidea's pollinators depends on pollinators entering several female figs. We hypothesized that it is in the interest of the plants to allow pollinators to re-emerge from figs on both male and female trees and that selection favors pollinator roaming because it increases their own reproductive success. Our manipulations of Blastophaga sp. pollinators in a Malaysian oil palm plantation confirmed that individual pollinators do routinely enter several figs of both sexes. Entering additional figs generated more seeds per pollinator on female trees and more pollinator offspring on male trees. Offspring sex ratios in subsequently entered figs were often less female-biased than in the first figs they entered, which reduced their immediate value to male trees because only female offspring carry their pollen. Small numbers of large seeds in female figs of epiphytic F. deltoidea may reflect constraints on overall female fig size, because pollinator exploitation depends on mutual mimicry between male and female figs.

Molecular mechanisms of mutualistic and antagonistic interactions in a plant-pollinator association.

Many insects metamorphose from antagonistic larvae into mutualistic adult pollinators, with reciprocal adaptation leading to specialized insect-plant associations. It remains unknown how such interactions are established at molecular level. Here we assemble high-quality genomes of a fig species, Ficus pumila var. pumila, and its specific pollinating wasp, Wiebesia pumilae. We combine multi-omics with validation experiments to reveal molecular mechanisms underlying this specialized interaction. In the plant, we identify the specific compound attracting pollinators and validate the function of several key genes regulating its biosynthesis. In the pollinator, we find a highly reduced number of odorant-binding protein genes and an odorant-binding protein mainly binding the attractant. During antagonistic interaction, we find similar chemical profiles and turnovers throughout the development of galled ovules and seeds, and a significant contraction of detoxification-related gene families in the pollinator. Our study identifies some key genes bridging coevolved mutualists, establishing expectations for more diffuse insect-pollinator systems.

Sky islands as foci for divergence of fig trees and their pollinators in southwest China.

The dynamics of populations and their divergence over time have shaped current levels of biodiversity and in the case of the "sky islands" of mountainous southwest (SW) China have resulted in an area of exceptional botanical diversity. Ficus tikoua is a prostrate fig tree subendemic to the area that displays unique intraspecific diversity, producing figs typical of different pollination modes in different parts of its range. By combining climate models, genetic variation in populations of the tree's obligate fig wasp pollinators and distributions of the different plant phenotypes, we examined how this unusual situation may have developed. We identified three genetically distinct groups of a single Ceratosolen pollinator species that have largely parapatric distributions. The complex topography of the region contributed to genetic divergence among the pollinators by facilitating geographical isolation and providing refugia. Migration along elevations in response to climate oscillations further enhanced genetic differentiation of the three pollinator groups. Their distributions loosely correspond to the distributions of the functionally significant morphological differences in the male figs of their host plants, but postglacial expansion of one group has not been matched by spread of its associated plant phenotype, possibly due to a major river barrier. The results highlight how interplay between the complex topography of the "sky island" complex and climate change has shaped intraspecies differentiation and relationships between the plant and its pollinator. Similar processes may explain the exceptional botanical diversity of SW China.

Loss of top-down biotic interactions changes the relative benefits for obligate mutualists.

The collapse of mutualisms owing to anthropogenic changes is contributing to losses of biodiversity. Top predators can regulate biotic interactions between species at lower trophic levels and may contribute to the stability of such mutualisms, but they are particularly likely to be lost after disturbance of communities. We focused on the mutualism between the fig tree Ficus microcarpa and its host-specific pollinator fig wasp and compared the benefits accrued by the mutualists in natural and translocated areas of distribution. Parasitoids of the pollinator were rare or absent outside the natural range of the mutualists, where the relative benefits the mutualists gained from their interaction were changed significantly away from the plant's natural range owing to reduced seed production rather than increased numbers of pollinator offspring. Furthermore, in the absence of the negative effects of its parasitoids, we detected an oviposition range expansion by the pollinator, with the use of a wider range of ovules that could otherwise have generated seeds. Loss of top-down control has therefore resulted in a change in the balance of reciprocal benefits that underpins this obligate mutualism, emphasizing the value of maintaining food web complexity in the Anthropocene.

Multiple parapatric pollinators have radiated across a continental fig tree displaying clinal genetic variation.

The ways that plant-feeding insects have diversified are central to our understanding of terrestrial ecosystems. Obligate nursery pollination mutualisms provide highly relevant model systems of how plants and their insect associates have diversified and the over 800 species of fig trees (Ficus) allow comparative studies. Fig trees can have one or more pollinating fig wasp species (Agaonidae) that breed within their figs, but factors influencing their number remain to be established. In some widely distributed fig trees, the plants form populations isolated by large swathes of sea, and the different populations are pollinated by different wasp species. Other Ficus species with continuous distributions may present genetic signatures of isolation by distance, suggesting more limited pollinator dispersal, which may also facilitate pollinator speciation. We tested the hypothesis that Ficus hirta, a species for which preliminary data showed genetic isolation by distance, would support numerous pollinator species across its range. Our results show that across its range F. hirta displays clinal genetic variation and is pollinated by nine parapatric species of Valisia. This is the highest number of pollinators reported to date for any Ficus species, and it is the first demonstration of the occurrence of parapatric pollinator species on a fig host displaying continuous genetic structure. Future comparative studies across Ficus species should be able to establish the plant traits that have driven the evolution of pollinator dispersal behaviour, pollinator speciation and host plant spatial genetic structure.

A new species of Silba (Diptera; Lonchaeidae) associated with figs.

A new species of Silba Macquart 1851, Silba ischnopoda sp. nov., is described from Thailand and Cambodia. The Thailand individuals were obtained from larvae found in mature male figs of the dioecious Ficus ischnopoda Miq. Comparisons are made with other similar Asian Silba species which have also been reared from the figs of Ficus species. Male figs support development of fig wasp larvae, not seeds, suggesting that the larvae of S. ischnopoda may be predaceous.

Chilocoris capensis n. sp., the first species of the genus Chilocoris Mayr, 1865 (Hemiptera: Heteroptera: Cydnidae) recorded in the Republic of South Africa with an annotated checklist of South African burrower bugs.

Chilocoris capensis n. sp. collected from fallen ripe figs of broom cluster fig Ficus sur Forsskål, 1775, the first burrower bug species of the genus Chilocoris Mayr, 1865 recorded in the Republic of South Africa, is described and compared with Chilocoris laevicollis Horváth, 1919, the morphologically most closely allied Afrotropical species. Additionally, an annotated checklist of burrower bug species recorded in the Republic of South Africa is provided. The known biology of Afrotropical Chilocoris species is briefly summarized.

Seasonality of Leaf and Fig Production in Ficus squamosa, a Fig Tree with Seeds Dispersed by Water.

The phenology of plants reflects selection generated by seasonal climatic factors and interactions with other plants and animals, within constraints imposed by their phylogenetic history. Fig trees (Ficus) need to produce figs year-round to support their short-lived fig wasp pollinators, but this requirement is partially de-coupled in dioecious species, where female trees only develop seeds, not pollinator offspring. This allows female trees to concentrate seed production at more favorable times of the year. Ficus squamosa is a riparian species whose dispersal is mainly by water, rather than animals. Seeds can float and travel in long distances. We recorded the leaf and reproductive phenology of 174 individuals for three years in Chiang Mai, Northern Thailand. New leaves were produced throughout the year. Fig production occurred year-round, but with large seasonal variations that correlated with temperature and rainfall. Female and male trees initiated maximal fig crops at different times, with production in female trees confined mainly to the rainy season and male figs concentrating fig production in the preceding months, but also often bearing figs continually. Ficus squamosa concentrates seed production by female plants at times when water levels are high, favouring dispersal by water, and asynchronous flowering within male trees allow fig wasps to cycle there, providing them with potential benefits by maintaining pollinators for times when female figs become available to pollinate.

Extremely high proportions of male flowers and geographic variation in floral ratios within male figs of Ficus tikoua despite pollinators displaying active pollen collection.

Most plants are pollinated passively, but active pollination has evolved among insects that depend on ovule fertilization for larval development. Anther-to-ovule ratios (A/O ratios, a coarse indicator of pollen-to-ovule ratios) are strong indicators of pollination mode in fig trees and are consistent within most species. However, unusually high values and high variation of A/O ratios (0.096-10.0) were detected among male plants from 41 natural populations of Ficus tikoua in China. Higher proportions of male (staminate) flowers were associated with a change in their distribution within the figs, from circum-ostiolar to scattered. Plants bearing figs with ostiolar or scattered male flowers were geographically separated, with scattered male flowers found mainly on the Yungui Plateau in the southwest of our sample area. The A/O ratios of most F. tikoua figs were indicative of passive pollination, but its Ceratosolen fig wasp pollinator actively loads pollen into its pollen pockets. Additional pollen was also carried on their body surface and pollinators emerging from scattered-flower figs had more surface pollen. Large amounts of pollen grains on the insects' body surface are usually indicative of a passive pollinator. This is the first recorded case of an actively pollinated Ficus species producing large amounts of pollen. Overall high A/O ratios, particularly in some populations, in combination with actively pollinating pollinators, may reflect a response by the plant to insufficient quantities of pollen transported in the wasps' pollen pockets, together with geographic variation in this pollen limitation. This suggests an unstable scenario that could lead to eventual loss of wasp active pollination behavior.

Insect responses to host plant provision beyond natural boundaries: latitudinal and altitudinal variation in a Chinese fig wasp community.

Many plants are grown outside their natural ranges. Plantings adjacent to native ranges provide an opportunity to monitor community assembly among associated insects and their parasitoids in novel environments, to determine whether gradients in species richness emerge and to examine their consequences for host plant reproductive success. We recorded the fig wasps (Chalcidoidea) associated with a single plant resource (ovules of Ficus microcarpa) along a 1200 km transect in southwest China that extended for 1000 km beyond the tree's natural northern range margin. The fig wasps included the tree's agaonid pollinator and other species that feed on the ovules or are their parasitoids. Phytophagous fig wasps (12 species) were more numerous than parasitoids (nine species). The proportion of figs occupied by fig wasps declined with increasing latitude, as did the proportion of utilized ovules in occupied figs. Species richness, diversity, and abundance of fig wasps also significantly changed along both latitudinal and altitudinal gradients. Parasitoids declined more steeply with latitude than phytophages. Seed production declined beyond the natural northern range margin, and at high elevation, because pollinator fig wasps became rare or absent. This suggests that pollinator climatic tolerances helped limit the tree's natural distribution, although competition with another species may have excluded pollinators at the highest altitude site. Isolation by distance may prevent colonization of northern sites by some fig wasps and act in combination with direct and host-mediated climatic effects to generate gradients in community composition, with parasitoids inherently more sensitive because of declines in the abundance of potential hosts.

Movements of genes between populations: are pollinators more effective at transferring their own or plant genetic markers?

The transfer of genes between populations is increasingly important in a world where pollinators are declining, plant and animal populations are increasingly fragmented and climate change is forcing shifts in distribution. The distances that pollen can be transported by small insects are impressive, as is the extensive gene flow between their own populations. We compared the relative ease by which small insects introduce genetic markers into their own and host-plant populations. Gene flow via seeds and pollen between populations of an Asian fig species were evaluated using cpDNA and nuclear DNA markers, and between-population gene flow of its pollinator fig wasp was determined using microsatellites. This insect is the tree's only pollinator locally, and only reproduces in its figs. The plant's pollen-to-seed dispersal ratio was 9.183-9.437, smaller than that recorded for other Ficus. The relative effectiveness of the pollinator at introducing markers into its own populations was higher than the rate it introduced markers into the plant's populations (ratio = 14 : 1), but given the demographic differences between plant and pollinator, pollen transfer effectiveness is remarkably high. Resource availability affects the dispersal of fig wasps, and host-plant flowering phenology here and in other plant-pollinator systems may strongly influence relative gene flow rates.

Spatial heterogeneity and host repression in fig-fig wasp mutualism.

It is generally believed that physical heterogeneity in common resource or evolutionary restraint can sufficiently prevent direct conflict between host and symbionts in mutualism systems. Our data on fig/fig wasp reciprocal mutualism (Ficus racemosa), however, show that structural barriers of female flowers or genetic constraints of pollinators previously hypothesized exist, but cannot sufficiently maintain the mutualism stability. The results show that a positive relationship between seed and wasp production could be maintained in warm season, which might be because of density dependence restraint among foundresses and their low oviposition and pollination efficiency, keeping common resource (female flowers) utilization unsaturated. Whilst, a negative correlation between wasp offspring and viable seed production was also observed in cold season, which might be that the increased oviposition and pollination efficiency maximized the common resource utilization. The fitness trade-off between fig and pollinator wasps is greatly affected by environmental or ecological variations. The local stability might result from temporal low exploitation efficiency of pollinators together with interference competition among pollinators. We suggest that host repression through the active regulation of bract closure, which can create interference competition among the foundresses and prevent extra more foundresses sequential entry in fruit cavities, would help the figs avoiding the cost of over-exploitation. This essentially takes the same role as sanctioning of cheating or competitive behaviors.

Phenological adaptations in Ficus tikoua exhibit convergence with unrelated extra-tropical fig trees.

Flowering phenology is central to the ecology and evolution of most flowering plants. In highly-specific nursery pollination systems, such as that involving fig trees (Ficus species) and fig wasps (Agaonidae), any mismatch in timing has serious consequences because the plants must balance seed production with maintenance of their pollinator populations. Most fig trees are found in tropical or subtropical habitats, but the dioecious Chinese Ficus tikoua has a more northerly distribution. We monitored how its fruiting phenology has adapted in response to a highly seasonal environment. Male trees (where fig wasps reproduce) had one to three crops annually, whereas many seed-producing female trees produced only one fig crop. The timing of release of Ceratosolen fig wasps from male figs in late May and June was synchronized with the presence of receptive figs on female trees, at a time when there were few receptive figs on male trees, thereby ensuring seed set while allowing remnant pollinator populations to persist. F. tikoua phenology has converged with those of other (unrelated) northern Ficus species, but there are differences. Unlike F. carica in Europe, all F. tikoua male figs contain male flowers, and unlike F. pumila in China, but like F. carica, it is the second annual generation of adult wasps that pollinate female figs. The phenologies of all three temperate fig trees generate annual bottlenecks in the size of pollinator populations and for female F. tikoua also a shortage of fig wasps that results in many figs failing to be pollinated.

Riparian Ficus tree communities: the distribution and abundance of riparian fig trees in northern Thailand.

Fig trees (Ficus) are often ecologically significant keystone species because they sustain populations of the many seed-dispersing animals that feed on their fruits. They are prominent components of riparian zones where they may also contribute to bank stability as well as supporting associated animals. The diversity and distributions of riparian fig trees in deciduous and evergreen forests in Chiang Mai Province, Northern Thailand were investigated in 2010-2012. To record the diversity and abundance of riparian fig trees, we (1) calculated stem density, species richness, and diversity indices in 20×50 m randomly selected quadrats along four streams and (2) measured the distances of individual trees from four streams to determine if species exhibit distinct distribution patterns within riparian zones. A total of 1169 individuals (from c. 4 ha) were recorded in the quadrats, representing 33 Ficus species (13 monoecious and 20 dioecious) from six sub-genera and about 70% of all the species recorded from northern Thailand. All 33 species had at least some stems in close proximity to the streams, but they varied in their typical proximity, with F. squamosa Roxb. and F. ischnopoda Miq the most strictly stream-side species. The riparian forests in Northern Thailand support a rich diversity and high density of Ficus species and our results emphasise the importance of fig tree within the broader priorities of riparian area conservation. Plans to maintain or restore properly functioning riparian forests need to take into account their significance.

Competitive exclusion among fig wasps achieved via entrainment of host plant flowering phenology.

Molecular techniques are revealing increasing numbers of morphologically similar but co-existing cryptic species, challenging the niche theory. To understand the co-existence mechanism, we studied phenologies of morphologically similar species of fig wasps that pollinate the creeping fig (F. pumila) in eastern China. We compared phenologies of fig wasp emergence and host flowering at sites where one or both pollinators were present. At the site where both pollinators were present, we used sticky traps to capture the emerged fig wasps and identified species identity using mitochondrial DNA COI gene. We also genotyped F. pumila individuals of the three sites using polymorphic microsatellites to detect whether the host populations were differentiated. Male F. pumila produced two major crops annually, with figs receptive in spring and summer. A small partial third crop of receptive figs occurred in the autumn, but few of the second crop figs matured at that time. Hence, few pollinators were available to enter third crop figs and they mostly aborted, resulting in two generations of pollinating wasps each year, plus a partial third generation. Receptive figs were produced on male plants in spring and summer, timed to coincide with the release of short-lived adult pollinators from the same individual plants. Most plants were pollinated by a single species. Plants pollinated by Wiebesia sp. 1 released wasps earlier than those pollinated by Wiebesia sp. 3, with little overlap. Plants occupied by different pollinators were not spatially separated, nor genetically distinct. Our findings show that these differences created mismatches with the flight periods of the other Wiebesia species, largely 'reserving' individual plants for the resident pollinator species. This pre-emptive competitive displacement may prevent long term co-existence of the two pollinators.