Exploring systematic biases, rooting methods and morphological evidence to unravel the evolutionary history of the genus Ficus (Moraceae).

Despite many attempts in the Sanger sequencing era, the phylogeny of fig trees remains unresolved, which limits our ability to analyze the evolution of key traits that may have contributed to their evolutionary and ecological success. We used restriction-site-associated DNA sequencing (c. 420 kb) and 102 morphological characters to elucidate the relationships between 70 species of Ficus. To increase phylogenetic information for higher-level relationships, we targeted conserved regions and assembled paired reads into long loci to enable the retrieval of homologous loci in outgroup genomes. We compared morphological and molecular results to highlight discrepancies and reveal possible inference bias. For the first time, we recovered a monophyletic subgenus Urostigma (stranglers) and a clade with all gynodioecious Ficus. However, we show, with a new approach based on iterative principal component analysis, that it is not (and will probably never be) possible to homogenize evolutionary rates and GC content for all taxa before phylogenetic inference. Four competing positions for the root of the molecular tree are possible. The placement of section Pharmacosycea as sister to other fig trees is not supported by morphological data and considered a result of a long-branch attraction artefact to the outgroups. Regarding morphological features and indirect evidence from the pollinator tree of life, the topology that divides Ficus into monoecious versus gynodioecious species appears most plausible. It seems most likely that the ancestor of fig trees was a freestanding tree and active pollination is inferred as the ancestral state, contrary to previous hypotheses. However, ambiguity remains on the ancestral breeding system. Despite morphological plasticity, we advocate restoring a central role to morphology in our understanding of the evolution of Ficus, as it can help detect systematic errors that appear more pronounced with larger molecular datasets.

Fig fruit ripening is regulated by the interaction between ethylene and abscisic acid.

Fleshy fruit ripening is typically regulated by ethylene in climacteric fruits and abscisic acid (ABA) in non-climacteric fruits. Common fig (Ficus carica) shows a dual-ripening mechanism, which is not fully understood. Here, we detected separate peaks of ethylene and ABA in fig fruits at the onset- and on-ripening stages, in conjunction with a sharp rise in glucose and fructose contents. In a newly-designed split-fruit system, exogenous ethylene failed to rescue fluridone-inhibited fruit ripening, whereas exogenous ABA rescued 2-amino-ethoxy-vinyl glycine (AVG)-inhibited fruit ripening. Transcriptome analysis revealed changes in the expression of genes key to both ABA and ethylene biosynthesis and perception during fig fruit ripening. At the de-greening stage, downregulation of FcACO2 or FcPYL8 retarded ripening, but downregulation of FcETR1/2 did not; unexpectedly, downregulation of FcAAO3 promoted ripening, but it inhibited ripening only before the de-greening stage. Furthermore, we detected an increase in ethylene emissions in the FcAAO3-RNAi ripening fruit and a decrease in ABA levels in the FcACO2-RNAi unripening fruit. Importantly, FcPYL8 can bind to ABA, suggesting that it functions as an ABA receptor. Our findings support the hypothesis that ethylene regulates the fig fruit ripening in an ABA-dependent manner. We propose a model for the role of the ABA-ethylene interaction in climacteric/non-climacteric processes.

Molecular phylogeny of the Ficus virens complex (Moraceae).

The closely related species present in the subgenera of Urostigma are challenging to classify due to the existence of overlapping morphological characteristics, which makes identification habitually problematic. It is still unresolved whether the species of the Ficus virens complex, which includes F. virens, F. middletonii, F. caulocarpa, F. concinna, and F. superba, are the same or distinct species due to the complexities in classification. To clarify the circumscription between the species and re-evaluate the taxonomical status, morphological characteristics were extensively examined; further, a phylogenetic reconstruction based on two DNA markers (ITS2 and trnH-psbA) in combination with morphological traits was carried out. The phylogenetic tree constructed using the combined morphology and DNA markers revealed that the five species should be demarcated as independent species. This study supports the importance of using both molecular and morphological data for efficient discrimination of species having high similarities. Further investigation into the species present in the subgenera Urostigma may provide additional information regarding the ancestral traits and its evolutionary history.

From dangerous branches to urban banyan: Facilitating aerial root growth of Ficus rubiginosa.

Large urban trees have many benefits. However, falling branches pose a serious hazard to both people and infrastructure. In several tree species, aerial roots grow down from branches to the ground. These roots are capable of thickening to support the branches, lessening the risk of tree failure. Unfortunately, in urban environments most aerial roots die before reaching the ground. Here, we report a new method for encouraging aerial roots to reach the ground, developed by the second-year botany class at UNSW Sydney. Our class tested three experimental treatments on aerial roots of Ficus rubiginosa Desf. ex Vent. (Port Jackson Fig)-PVC pipes filled with sphagnum moss, PVC pipes filled with potting mix, and PVC pipes filled with sphagnum moss and topped with funnels to catch extra rainwater. All three treatments significantly improved aerial root growth, with 26 of the 30 (87%) treatment roots reaching the ground after one year compared to 0 of the 10 control roots. Our method was successful for roots up to 3 m above the ground, suggesting the potential growth rate of aerial roots is substantial when conditions are favourable. Our novel approach is an attractive and cost-effective alternative to slings and other artificial supports. This project is an example of using undergraduate practical classes to teach science while simultaneously addressing important real-world problems.

A 3D study of the relationship between leaf vein structure and mechanical function.

We investigate the structures and mechanical properties of leaf midribs of Ficus microcarpa and Prunus dulcis, which deposit calcium oxalate crystals, and of Olea europaea midribs which contain no mineral deposits, but do contain lignified fibers. The midrib mechanical performance contributes to the leaf's ability to maintain a flat conformation for light harvesting and to efficiently reconfigure to reduce wind drag. We use a novel approach involving 3D visualization of the vein structure during mechanical load. This involves the use of customized mechanical loading devices that fit inside a microCT chamber. We show that the elastic, compression and torsional moduli of the midribs of leaves from the 3 species examined vary significantly. We also observed different modes of fracture and buckling of the leaves during compression. We assess the contributions of the calcium oxalate crystals to the mechanical and fracture properties. In F. microcarpa midrib linear arrays of calcium oxalate crystals contribute to resisting the bending, in contrast to P. dulcis leaves, where the calcium oxalate crystals do not resist bending. In both F. microcarpa and P. dulcis isolated calcium oxalate crystals enable high torsional compliance. The integrated microCT - mechanical testing approach could be used to investigate the structure-mechanics relationships in other complex biological samples. STATEMENT OF SIGNIFICANCE: Leaves need to maintain a flat conformation for light harvesting, but they also need to efficiently reconfigure to reduce wind drag. The leaf central vein (midrib) is a key structural component for leaf mechanicss. 3D visualization of the vein structure under mechanical loads showed that veins can be stiffened by reinforcement units composed of calcium oxalates crystals and lignin. The stiffening units can influence the bending and fracture properties of the midribs, and can contribute to determine if buckling will occur during folding. Mineral stiffening elements could be a widespread strategy to reinforce leaf veins and other biological structures. This structural-mechanical approach could be used to study other complex biological samples.

Plant geographic phenotypic variation drives diversification in its associated community of a phytophagous insect and its parasitoids.

BACKGROUND: While the communities constituted by phytophageous insects and their parasites may represent half of all terrestrial animal species, understanding their diversification remains a major challenge. A neglected idea is that geographic phenotypic variation in a host plant may lead to heterogeneous evolutionary responses of the different members of the associated communities. This could result in diversification on a host plant by ecological speciation in some species, leading to geographic variation in community composition. In this study we investigated geographic variation of inflorescence receptacle size in a plant, Ficus hirta, and how the hymenopteran community feeding in the inflorescences has responded. Our predictions were: 1) Inflorescence size variation affects wasp species differently depending on how they access oviposition sites. 2) In some affected lineages of wasps, we may observe vicariant, parapatric species adapted to different inflorescence sizes. RESULTS: We show that fig (the enclosed inflorescence of Ficus) wall thickness varies geographically. The fig-entering pollinating wasp was not affected, while the parasites ovipositing through the fig wall were. Two parapatric species of Philotrypesis, exhibiting strikingly different ovipositor lengths, were recorded. One species of Sycoscapter was also present, and it was restricted, like the shorter-ovipositor Philotrypesis, to the geographic zone where fig walls were thinner. CONCLUSIONS: Previous work on fig wasps suggested that parapatric geographic ranges among congenerics were due to adaptation to variation in abiotic factors, complemented by interspecific competition. Our results show that parapatric ranges may also result from adaptation to variation in biotic factors. Within an insect community, differences among species in their response to geographic phenotypic variation of their host plant may result in geographically heterogeneous community structure. Such heterogeneity leads to heterogeneous interaction networks among sites. Our results support the hypothesis that plant geographic phenotypic variation can be a driver of diversification in associated insect communities, and can complement other diversification processes.

Diversity of fig glands is associated with nursery mutualism in fig trees.

PREMISE OF THE STUDY: Fig trees (Moraceae) have remarkable enclosed inflorescences called figs or syconia. The flowers are pollinated by host-specific fig wasps that enter the fig to lay their eggs. This nursery pollination system is one of the most studied of tropical mutualism interactions, but the source of the volatiles that attract fig wasps to their specific host figs has not been confirmed. The fragrance is the basis of host selection and, therefore, of reproductive isolation among sympatric Ficus species. This study locates and characterizes the glands likely to be responsible for pollinator attraction and also protection from herbivory in the figs of nine Ficus species representing all the major lineages within the genus. METHODS: Figs with receptive pistillate flowers were examined using light and scanning electron microscopy. Tests for histolocalization of substances were employed to detect glandular activity throughout the figs. KEY RESULTS: A great diversity of glands is found throughout the fig, and for the first time, the sites producing fragrances are identified. Scent glands are present on the ostiolar bracts and the outer layers of the fig receptacle. Laticifers and phenolic-producing idioblasts, epidermis, and trichomes associated with fig protection occur on the ostiolar bracts, the fig receptacle, and floral tissues. CONCLUSIONS: The volatiles produced by glands on the ostiolar bracts are candidate sources for the long-distance attraction of pollinator fig wasps. Scent glands on the outer layers of the receptacle may also play a role in chemical perception of the figs or may be related to their protection. The high cost to the plants if the figs are eaten and the temperature conditions required for nursery pollination are likely the factors that led to the selection of phenolic glands and laticifers during the group's evolution.

Leaf anatomy and histochemistry of three species of Ficus sect. Americanae supported by light and electron microscopy.

In this work the leaf anatomy of three species of Ficus section Americanae (Miq.) Miq. from Brazil, whose leaves and latex are used in folk medicine is reported. The work was carried out using light and scanning electron microscopy in order to characterize these species and to evaluate their taxonomic significance, and also contribute to the quality control of their ethnodrugs. The three species (Ficus cyclophylla, Ficus elliotiana, and Ficus caatingae) showed hypostomatic leaves, anomocytic stomata, straight epidermal cell outlines, and a dorsiventral mesophyll. Some micro-morphological characters such as density and distribution of epicuticular waxes, glandular trichomes, the length and width of stomata, as well as the palisade of mesophyll and petiole outlines proved to be the most useful and distinctive characters for the separation of species. These may contribute as additional support for the taxonomy of the section and for the quality control of their ethnodrugs.

LEAF GUI: analyzing the geometry of veins and areoles using image segmentation algorithms.

The Leaf Extraction and Analysis Framework Graphical User Interface (LEAF GUI) software is designed for biologists who wish to analyze the structure of vessel bundles (veins) in leaves. The software enables users to extract descriptive statistics on the dimensions and positions of leaf veins and areoles by utilizing a series of thresholding, cleaning, and segmentation algorithms applied to images of leaf veins. The resulting statistics for the dimensions of individual veins and the areoles they surround can then be used to evaluate numerous hypotheses regarding the structure and function of leaf veins.

Natural foliar variegation without costs? The case of Begonia.

BACKGROUND AND AIMS: Foliar variegation is recognized as arising from two major mechanisms: leaf structure and pigment-related variegation. Begonia has species with a variety of natural foliar variegation patterns, providing diverse examples of this phenomenon. The aims of this work are to elucidate the mechanisms underlying different foliar variegation patterns in Begonia and to determine their physiological consequences. METHODS: Six species and one cultivar of Begonia were investigated. Light and electron microscopy revealed the leaf structure and ultrastructure of chloroplasts in green and light areas of variegated leaves. Maximum quantum yields of photosystem II were measured by chlorophyll fluorescence. Comparison with a cultivar of Ficus revealed key features distinguishing variegation mechanisms. KEY RESULTS: Intercellular space above the chlorenchyma is the mechanism of variegation in these Begonia. This intercellular space can be located (a) below the adaxial epidermis or (b) below the adaxial water storage tissue (the first report for any taxa), creating light areas on a leaf. In addition, chlorenchyma cell shape and chloroplast distribution within chlorenchyma cells differ between light and green areas. Chloroplasts from both areas showed dense stacking of grana and stroma thylakoid membranes. The maximum quantum yield did not differ significantly between these areas, suggesting minimal loss of function with variegation. However, the absence of chloroplasts in light areas of leaves in the Ficus cultivar led to an extremely low quantum yield. CONCLUSIONS: Variegation in these Begonia is structural, where light areas are created by internal reflection between air spaces and cells in a leaf. Two forms of air space structural variegation occur, distinguished by the location of the air spaces. Both forms may have a common origin in development where dermal tissue becomes loosely connected to mesophyll. Photosynthetic functioning is retained in light areas, and these areas do not include primary veins, potentially limiting the costs of variegation.

Exchange of hosts: can agaonid fig wasps reproduce successfully in the figs of non-host Ficus?

In the obligate mutualism between figs (Ficus) and their specific pollinators (Chalcidoidea, Agaonidae), each species of fig wasp typically reproduces in figs of a single host species. Host specificity is maintained largely because pollinators are attracted to tree-specific volatiles released from their host figs, but whether the wasps can reproduce if they enter figs of non-host species is unclear. We investigated the reproductive success of Ceratosolen emarginatus (associated with Ficus auriculata) and Ceratosolen sp. (associated with F. hainanensis) in atypical hosts by experimentally introducing foundresses into host and non-host figs. F. auriculata figs entered by Ceratosolen sp. were more likely to abort than if entered by C. emarginatus, but abortion of F. hainanensis figs was not affected by pollinator species. Single C. emarginatus foundresses produced more but smaller offspring in F. hainanensis than in their normal host. Conversely Ceratosolen sp. produced fewer but larger offspring in F. auriculata than in their normal host, probably as a result of having longer to develop. Mean style length differences, relative to the lengths of the wasps' ovipositors, may have dictated the number of offspring produced, with oviposition made easier by the shorter styles in F. hainanensis figs. Our results imply that, in addition to morphological constraints and tree-specific volatiles, reduced reproductive success in atypical hosts can be another factor maintaining host specificity, but for other species only behavioural changes are required for host switching to occur.

Morphological phylogenetic analysis of seven varieties of Ficus deltoidea Jack from the Malay Peninsula of Malaysia.

This study is the first report to suggest a morphological phylogenetic framework for the seven varieties of Ficus deltoidea Jack (Ficus: Moraceae) from the Malay Peninsula of Malaysia. Several molecular-based classifications on the genus Ficus had been proposed, but neither had discussed the relationship between seven varieties of F. deltoidea to its allies nor within the varieties. The relationship between seven varieties of F. deltoidea is still debated due to the extreme morphological variabilities and ambiguous boundaries between taxa. Thus, the correct identification of these varieties is important as several morphological characters are variety-specific. To test the monophyly and further resolved the relationship in F. deltoidea, a morphological phylogenetic analysis was conducted based on herbarium specimens representing the seven varieties of F. deltoidea that were collected from the Malay Peninsula of Malaysia, by using related species of the genus Ficus; F. grossularioides, F. ischnopoda and F. oleifolia as the outgroups. Parsimony and neighbour-joining analyses indicated that F. deltoidea is monophyletic, in that the seven varieties of F. deltoidea nested into two clades; clade subspecies deltoidea (var. deltoidea, var. bilobata, var. angustifolia, var. kunstleri and var. trengganuensis) and clade subspecies motleyana (var. intermedia and var. motleyana).

Ancient fig wasps indicate at least 34 Myr of stasis in their mutualism with fig trees.

Fig wasps and fig trees are mutually dependent, with each of the 800 or so species of fig trees (Ficus, Moraceae) typically pollinated by a single species of fig wasp (Hymenoptera: Agaonidae). Molecular evidence suggests that the relationship existed over 65 Ma, during the Cretaceous. Here, we record the discovery of the oldest known fossil fig wasps, from England, dated at 34 Ma. They possess pollen pockets that contain fossil Ficus pollen. The length of their ovipositors indicates that their host trees had a dioecious breeding system. Confocal microscopy and scanning electron microscopy reveal that the fossil female fig wasps, and more recent species from Miocene Dominican amber, display the same suite of anatomical characters associated with fig entry and pollen-carrying as modern species. The pollen is also typical of modern Ficus. No innovations in the relationship are discernible for the last tens of millions of years.

Elucidating genetic relationships, diversity and population structure among the Turkish female figs.

A collection of 96 female Turkish fig (Ficus carica L.) accessions was studied to elucidate genetic structure and estimate diversity and genetic similarity distribution among the female figs present in Turkish genetic resources, using 157 molecular genome markers including 129 sequence-related amplified polymorphisms, 21 random amplified polymorphic DNAs, and 7 simple-sequence repeats. The plant samples mainly included Turkish fig collections selected throughout the country over the course of a half-century. Neighbor-joining analysis revealed continuous dissimilarity range, and it was difficult to classify figs into distinct groups. The principle component analysis produced similar results. The analysis of molecular variance indicated that 95 and 93% of genetic variation were explained by within geographic origins and similar fruit rind color, respectively. Sub-structuring Bayesian analysis assigned the 96 female figs into four sub-populations, and indicated that they were highly related. The corrected allelic pairwise distances among the six geographic origins were less than 5%. This study suggests that geography- and color-based groups were not genetically distinct among the Turkish figs.

Oxidative stress enzymes in Ficus religiosa L.: biochemical, histochemical and anatomical evidences.

In order to unveil the reasons behind the successful survival of Ficus religiosa L. grown under normal and adverse habitats (AH), i.e., on concrete roof tops were subjected to biochemical, histochemical and physiological studies with a focus on reactive oxygen species (ROS) and oxidative stress enzymes (OSE). The specific objectives were: to localize the OSE, peroxidase (POX) and catalase (CAT); to localize and quantify the main ROS, hydrogen peroxide (H(2)O(2)); to estimate the activities of POX, CAT and glycolate oxidase (GO); and to study the diurnal variations in stomatal activity by scanning electron microscopy (SEM). From the results, plants grown in AH showed 55% higher H(2)O(2) production with about 30% increase in POX activity. Of the three substrates tested for POX activity (guaiacol, ascorbate and o-dianisidine), o-dianisidine was found as the preferred substrate of F. religiosa POX with about 7-fold more activity over its counterparts. Cytosolic POX activity showed 11-fold increase over cell wall bound POX. Similarly, CAT activity in specimens from AH showed about 2-fold increase during day time. The physiological interaction between CAT and its substrate H(2)O(2) in the plant was determined by quantifying H(2)O(2) and assaying the CAT, in which CAT showed 4-fold increases in activity, especially during night. F. religiosa has higher amount of H(2)O(2) deposition during night than day time, which was in correlation high CAT activity during night, coupled with scotoactive opening of stomata as shown by the SEM images. Moreover, GO did not show much habitat-dependent variation. In toto, F. religiosa grown in AH showed elevated production of ROS and their scavenging OSE, which is the direct evidence for drought stress and also giving an insight into its evolution and ecological niche.

Correlated evolution of fig size and color supports the dispersal syndromes hypothesis.

The influence of seed dispersers on the evolution of fruit traits remains controversial, largely because most studies have failed to account for phylogeny and or have focused on conservative taxonomic levels. Under the hypothesis that fruit traits have evolved in response to different sets of selective pressures by disparate types of seed dispersers (the dispersal syndromes hypothesis), we test for two dispersal syndromes, defined as groups of fruit traits that appear together more often than expected by chance. (1) Bird syndrome fruits are brightly colored and small, because birds have acute color vision, and commonly swallow fruits whole. (2) Mammal syndrome fruits are dull-colored and larger on average than bird syndrome fruits, because mammals do not rely heavily on visual cues for finding fruits, and can eat fruits piecemeal. If, instead, phylogenetic inertia determines the co-occurrence of fruit size and color, we will observe that specific combinations of size and color evolved in a small number of ancestral species. We performed a comparative analysis of fruit traits for 64 species of Ficus (Moraceae), based on a phylogeny we constructed using nuclear ribosomal DNA. Using a concentrated changes test and assuming fruit color is an independent variable, we found that small-sized fruits evolve on branches with red and purple figs, as predicted by the dispersal syndromes hypothesis. When using diameter as the independent variable, results vary with the combination of algorithms used, which is discussed in detail. A likelihood ratio test confirms the pattern found with the concentrated changes test using color as the independent variable. These results support the dispersal syndromes hypothesis.

Food plant diversity as broad-scale determinant of avian frugivore richness.

The causes of variation in animal species richness at large spatial scales are intensively debated. Here, we examine whether the diversity of food plants, contemporary climate and energy, or habitat heterogeneity determine species richness patterns of avian frugivores across sub-Saharan Africa. Path models indicate that species richness of Ficus (their fruits being one of the major food resources for frugivores in the tropics) has the strongest direct effect on richness of avian frugivores, whereas the influences of variables related to water-energy and habitat heterogeneity are mainly indirect. The importance of Ficus richness for richness of avian frugivores diminishes with decreasing specialization of birds on fruit eating, but is retained when accounting for spatial autocorrelation. We suggest that a positive relationship between food plant and frugivore species richness could result from niche assembly mechanisms (e.g. coevolutionary adaptations to fruit size, fruit colour or vertical stratification of fruit presentation) or, alternatively, from stochastic speciation-extinction processes. In any case, the close relationship between species richness of Ficus and avian frugivores suggests that figs are keystone resources for animal consumers, even at continental scales.

[Leaf anatomy of the mosaic ficus benjamina cv. Starlight and interaction of source and sink chimera components].

Leaf anatomy was studied in the mosaic Ficus benjamina cv. Starlight and non-chimeric Ficus benjamina cv. Daniel. The number of chloroplasts in a white, chlorophyll-deficient tissue declines as compared to the green tissue. However, their functional activity is retained. The leaf of the mosaic F. benjamina contains two or, sometimes, three subepidermal layers. Mesophyll forms one layer in the green and white parts of leaf palisade and one white and one green layer in the transitional zone (edge). In the transitional zone, green spongy mesophyll is located between two white spongy layers and the proportion of photosynthesizing cells varies. In cv. Daniel, there are two subepidermal layers and one layer of columnar mesophyll cells. According to the morphometry data, the proportion of white zone in the leaf correlates with the leaf position in the whole shoot: the higher the branch order, the larger the proportion of white zone. The total leaf area depends also on its position in the shoot. No such correlation was found in non-chimeric F. benjamina cv. Daniel. In the mosaic chimera, the source-sink status appears to depend on the leaf position in the shoot. Experiments with individual shoots of the same order and elimination of all lateral shoots have shown that the proportion of white zone in new leaves on the shoot increases with the total area of green zone. Thus, the area of assimilating shoot surface affects the formation of leaves in the meristem. A hypothesis was put forward that the source-sink state affects the ratio of green and white parts in the leaf primordium. Products of photosynthesis (carbohydrates) are a possible metabolic signal affecting the meristem. It cannot be excluded as well that the hormonal state undergoes changes in the chimeric plant.

Early domesticated fig in the Jordan Valley.

It is generally accepted that the fig tree was domesticated in the Near East some 6500 years ago. Here we report the discovery of nine carbonized fig fruits and hundreds of drupelets stored in Gilgal I, an early Neolithic village, located in the Lower Jordan Valley, which dates to 11,400 to 11,200 years ago. We suggest that these edible fruits were gathered from parthenocarpic trees grown from intentionally planted branches. Hence, fig trees could have been the first domesticated plant of the Neolithic Revolution, which preceded cereal domestication by about a thousand years.