High genetic diversity and low population differentiation of a medical plant Ficus hirta Vahl., uncovered by microsatellite loci: implications for conservation and breeding.

BACKGROUND: Wuzhimaotao (Radix Fici Hirtae) originates from the dry root of Ficus hirta (Moraceae), which is widely known as a medical and edible plant distributed in South China. As the increasing demand for Wuzhimaotao, the wild F. hirta has been extremely reduced during the past years. It is urgent to protect and rationally develop the wild resources of F. hirta for its sustainable utilization. However, a lack of genetic background of F. hirta makes it difficult to plan conservation and breeding strategies for this medical plant. In the present study, a total of 414 accessions of F. hirta from 7 provinces in southern China were evaluated for the population genetics using 9 polymorphic SSR markers. RESULTS: A mean of 17.1 alleles per locus was observed. The expected heterozygosity (He) varied from 0.142 to 0.861 (mean = 0.706) in nine SSR loci. High genetic diversity (He = 0.706, ranged from 0.613 to 0.755) and low genetic differentiation among populations (G'ST = 0.147) were revealed at population level. In addition, analysis of molecular variance (AMOVA) indicated that the principal molecular variance existed within populations (96.2%) was significantly higher than that among populations (3.8%). Meanwhile, the three kinds of clustering methods analysis (STRUCTURE, PCoA and UPGMA) suggested that the sampled populations were clustered into two main genetic groups (K = 2). Mantel test showed a significant correlation between geographic and genetic distance among populations (R2 = 0.281, P < 0.001). Pollen flow, seed flow and/or geographical barriers might be the main factors that formed the current genetic patterns of F. hirta populations. CONCLUSIONS: This is a comprehensive study of genetic diversity and population structure of F. hirta in southern China. We revealed the high genetic diversity and low population differentiation in this medicinal plant and clarified the causes of its current genetic patterns. Our study will provide novel insights into the exploitation and conservation strategies for F. hirta.

Genome-wide analysis of JAZ family genes expression patterns during fig (Ficus carica L.) fruit development and in response to hormone treatment.

BACKGROUND: Jasmonate-ZIM domain (JAZ) repressors negatively regulate signal transduction of jasmonates, which regulate plant development and immunity. However, no comprehensive analysis of the JAZ gene family members has been done in the common fig (Ficus carica L.) during fruit development and hormonal treatment. RESULTS: In this study, 10 non-redundant fig JAZ family genes (FcJAZs) distributed on 7 chromosomes were identified in the fig genome. Phylogenetic and structural analysis showed that FcJAZ genes can be grouped into 5 classes. All the classes contained relatively complete TIFY and Jas domains. Yeast two hybrid (Y2H) results showed that all FcJAZs proteins may interact with the identified transcription factor, FcMYC2. Tissue-specific expression analysis showed that FcJAZs were highly expressed in the female flowers and roots. Expression patterns of FcJAZs during the fruit development were analyzed by RNA-Seq and qRT-PCR. The findings showed that, most FcJAZs were significantly downregulated from stage 3 to 5 in the female flower, whereas downregulation of these genes was observed in the fruit peel from stage 4 to 5. Weighted-gene co-expression network analysis (WGCNA) showed the expression pattern of FcJAZs was correlated with hormone signal transduction and plant-pathogen interaction. Putative cis-elements analysis of FcJAZs and expression patterns of FcJAZs which respond to hormone treatments revealed that FcJAZs may regulate fig fruit development by modulating the effect of ethylene or gibberellin. CONCLUSIONS: This study provides a comprehensive analysis of the FcJAZ family members and provides information on FcJAZs contributions and their role in regulating the common fig fruit development.

Differentiation of Ficus deltoidea varieties and chemical marker determination by UHPLC-TOFMS metabolomics for establishing quality control criteria of this popular Malaysian medicinal herb.

BACKGROUND: Ficus deltoidea Jack (Moraceae) is a plant used in Malaysia for various diseases including as a supplement in diabetes management. Morphology distinction of the 7 main varieties (var. angustifolia, var. bilobata, var. deltoidea, var. intermedia, var. kunstleri, var. motleyana and var. trengganuensis) is challenging due to the extreme leaf heterophylly and unclear varietal boundaries, making it difficult for quality control of F. deltoidea products. OBJECTIVE: We aimed to compare the phytochemical composition of 7 varieties growing in different conditions at various geographical locations. We also aimed to establish the quality control markers for the authentication of these varieties. METHODS: We applied untargeted UHPLC-TOFMS metabolomics to discriminate 100 leaf samples of F. deltoidea collected from 6 locations in Malaysia. A genetic analysis on 21 leaf samples was also performed to validate the chemotaxonomy differentiation. RESULTS: The PCA and HCA analysis revealed the existence of 3 chemotypes based on the differentiation in the flavonoid content. The PLS-DA analysis identified 15 glycosylated flavone markers together with 1 furanocoumarin. These markers were always consistent for the respective varieties, regardless of the geographical locations and growing conditions. The chemotaxonomy differentiation was in agreement with the DNA sequencing. In particular, var. bilobata accession which showed divergent morphology was also differentiated by the chemical fingerprints and genotype. CONCLUSION: Chemotype differentiation based on the flavonoid fingerprints along with the proposed markers provide a powerful identification tool to complement morphology and genetic analyses for the quality control of raw materials and products from F. deltoidea.

Spatial Scales of Genetic Structure in Free-Standing and Strangler Figs (Ficus, Moraceae) Inhabiting Neotropical Forests.

Wind-borne pollinating wasps (Agaonidae) can transport fig (Ficus sp., Moraceae) pollen over enormous distances (> 100 km). Because of their extensive breeding areas, Neotropical figs are expected to exhibit weak patterns of genetic structure at local and regional scales. We evaluated genetic structure at the regional to continental scale (Panama, Costa Rica, and Peru) for the free-standing fig species Ficus insipida. Genetic differentiation was detected only at distances > 300 km (Jost´s Dest = 0.68 ± 0.07 & FST = 0.30 ± 0.03 between Mesoamerican and Amazonian sites) and evidence for phylogeographic structure (RST>>permuted RST) was only significant in comparisons between Central and South America. Further, we assessed local scale spatial genetic structure (SGS, d ≤ 8 km) in Panama and developed an agent-based model parameterized with data from F. insipida to estimate minimum pollination distances, which determine the contribution of pollen dispersal on SGS. The local scale data for F. insipida was compared to SGS data collected for an additional free-standing fig, F. yoponensis (subgenus Pharmacosycea), and two species of strangler figs, F. citrifolia and F. obtusifolia (subgenus Urostigma) sampled in Panama. All four species displayed significant SGS (mean Sp = 0.014 ± 0.012). Model simulations indicated that most pollination events likely occur at distances > > 1 km, largely ruling out spatially limited pollen dispersal as the determinant of SGS in F. insipida and, by extension, the other fig species. Our results are consistent with the view that Ficus develops fine-scale SGS primarily as a result of localized seed dispersal and/or clumped seedling establishment despite extensive long-distance pollen dispersal. We discuss several ecological and life history factors that could have species- or subgenus-specific impacts on the genetic structure of Neotropical figs.

Structure and Principal Components Analyses Reveal an Intervarietal Fusion in Malaysian Mistletoe Fig (Ficus deltoidea Jack) Populations.

Genetic structure and biodiversity of the medicinal plant Ficus deltoidea have rarely been scrutinized. To fill these lacunae, five varieties, consisting of 30 F. deltoidea accessions were collected across the country and studied on the basis of molecular and morphological data. Molecular analysis of the accessions was performed using nine Inter Simple Sequence Repeat (ISSR) markers, seven of which were detected as polymorphic markers. ISSR-based clustering generated four clusters supporting the geographical distribution of the accessions to some extent. The Jaccard's similarity coefficient implied the existence of low diversity (0.50-0.75) in the studied population. STRUCTURE analysis showed a low differentiation among the sampling sites, while a moderate varietal differentiation was unveiled with two main populations of F. deltoidea. Our observations confirmed the occurrence of gene flow among the accessions; however, the highest degree of this genetic interference was related to the three accessions of FDDJ10, FDTT16 and FDKT25. These three accessions may be the genetic intervarietal fusion points of the plant's population. Principal Components Analysis (PCA) relying on quantitative morphological characteristics resulted in two principal components with Eigenvalue >1 which made up 89.96% of the total variation. The cluster analysis performed by the eight quantitative characteristics led to grouping the accessions into four clusters with a Euclidean distance ranged between 0.06 and 1.10. Similarly, a four-cluster dendrogram was generated using qualitative traits. The qualitative characteristics were found to be more discriminating in the cluster and PCA analyses, while ISSRs were more informative on the evolution and genetic structure of the population.

Nuclear and chloroplast DNA phylogeography of Ficus hirta: obligate pollination mutualism and constraints on range expansion in response to climate change.

This study uses a phylogeographic approach to investigate how interspecific interactions in an obligate pollination mutualism enhance or constrain dispersal and the range distributions of species through time. Fifteen populations of Ficus hirta, a bird-dispersed fig pollinated by a species-specific fig wasp, were sampled from Thailand to the northern limits of the tropical forest in China. These populations were assayed for six nuclear microsatellite loci and two intergenic chloroplast DNA sequences. Analyses of range expansion and genetic clustering indicated a relatively slow rate of range expansion from two or more southern glacial refugia. Low nuclear differentiation, combined with high interpopulation differentiation, and phylogeographic structuring of chloroplast variation indicated that seed dispersal has had a greater constraint than obligate interactions with fig wasps on the rate of post-glacial range expansion. This study is the first to investigate the phylogeographic history of a widely distributed southeast Asian tropical plant whose distribution extends to the northern limits of tropical forest habitat in China. It is also the first study of Ficus utilizing molecular data to evaluate whether species-specific pollination is a limitation or an aid to range expansion in response to climate change.

cDNA cloning of a novel gene codifying for the enzyme lycopene ß-cyclase from Ficus carica and its expression in Escherichia coli.

Lycopene beta-cyclase (ß-LCY) is the key enzyme that modifies the linear lycopene molecule into cyclic ß-carotene, an indispensable carotenoid of the photosynthetic apparatus and an important source of vitamin A in human and animal nutrition. Owing to its antioxidant activity, it is commercially used in the cosmetic and pharmaceutical industries, as well as an additive in foodstuffs. Therefore, ß-carotene has a large share of the carotenoidic market. In this study, we used reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE)-PCR to obtain and clone a cDNA copy of the gene Lyc-ß from Ficus carica (Lyc-ß Fc), which codes for the enzyme lycopene ß-cyclase (ß-LCY). Expression of this gene in Escherichia coli produced a single polypeptide of 56 kDa of weight, containing 496 amino acids, that was able to cycle both ends of the lycopene chain. Amino acid analysis revealed that the protein contained several conserved plant cyclase motifs. ß-LCY activity was revealed by heterologous complementation analysis, with lycopene being converted to ß-carotene as a result of the enzyme's action. The ß-LCY activity of the expressed protein was confirmed by high-performance liquid chromatography (HPLC) identification of the ß-carotene. The lycopene to ß-carotene conversion rate was 90%. The experiments carried out in this work showed that ß-LYC is the enzyme responsible for converting lycopene, an acyclic carotene, to ß-carotene, a bicyclic carotene in F. carica. Therefore, by cloning and expressing ß-LCY in E. coli, we have obtained a new gene for ß-carotene production or as part of the biosynthetic pathway of astaxanthin. So far, this is the first and only gene of the carotenoid pathway identified in F. carica.

Reconciling field observations of dispersal with estimates of gene flow.

Montgomery Slatkin, in a classic article concerning the role of gene flow in determining population genetic structure in natural populations noted an inconsistency between observations made in the field of limited dispersal of weakly flying insect and population genetic analyses of genetic structure which revealed extensive gene flow among populations. This phenomenon was subsequently termed Slatkin's Paradox. In this issue, Yu et al. (2010) provide an example of Slatkin's Paradox in a study of seed and pollen gene flow in the dioecious understory fig Ficus hirta in southern Asia. Given multiple field observations that showed the low vagility of fig wasp pollinators of dioecious figs and the high movement capacity of its vertebrate seed dispersers, the authors expected to see higher levels of gene movement of seed versus pollen. They compared neutral genetic differentiation across 15 populations separated by >2500km at six nuclear microsatellite and two chloroplast loci and found that F. hirta shows an order of magnitude higher level of gene flow of pollen relative to seeds that challenges observations of limited dispersal of fig wasps that pollinate dioecious figs. They propose broadening the application of Slatkin's Paradox beyond insects to include situations where an incongruity exists between ecological measures of low dispersal and high levels of effective gene flow.

Slatkin's Paradox: when direct observation and realized gene flow disagree. A case study in Ficus.

In flying insects, there is frequently a lack of congruence between empirical estimates of local demographic parameters and the prediction that differentiation between populations should decrease with increasing dispersal, a puzzling phenomenon known as Slatkin's Paradox. Here, we generalize Slatkin's Paradox to other taxa, drawing from available information on dispersal to predict the relative importance of pollen vs. seed migration in structuring broad-scale patterns of genetic variation in Ficus hirta, a dioecious fig whose pollen is dispersed by minute, species-specific fig wasps and whose seeds are disseminated by a variety of vagile vertebrates (especially bats and birds). Local-scale observational and genetic studies of dioecious understory figs suggest comparable rates of pollen and seed migration. In contrast, we found unusually low nuclear differentiation (F(ST) =0.037, R(ST) =0.074) and high chloroplast differentiation (G(ST) =0.729, N(ST) =0.798) among populations separated by up to 2850km, leading us to reject the hypothesis of equal pollen and seed migration rates and to obtain an equilibrium estimate of the ratio of pollen to seed migration of r=16.2-36.3. We reconcile this example of Slatkin's Paradox with previously published data for dioecious figs and relate it to the picture of exceptionally long-distance wasp-mediated pollen dispersal that is emerging for large monoecious fig trees. More generally, we argue that Slatkin's Paradox is a general phenomenon and suggest it may be common in plants and animals.

Host sanctions and pollinator cheating in the fig tree-fig wasp mutualism.

Theory predicts that mutualisms should be vulnerable to invasion by cheaters, yet mutualistic interactions are both ancient and diverse. What prevents one partner from reaping the benefits of the interaction without paying the costs? Using field experiments and observations, we examined factors affecting mutualism stability in six fig tree-fig wasp species pairs. We experimentally compared the fitness of wasps that did or did not perform their most basic mutualistic service, pollination. We found host sanctions that reduced the fitness of non-pollinating wasps in all derived, actively pollinated fig species (where wasps expend time and energy pollinating), but not in the basal, passively pollinated fig species (where wasps do not). We further screened natural populations of pollinators for wasp individuals that did not carry pollen ('cheaters'). Pollen-free wasps occurred only in actively pollinating wasp species, and their prevalence was negatively correlated with the sanction strength of their host species. Combined with previous studies, our findings suggest that (i) mutualisms can show coevolutionary dynamics analogous to those of 'arms races' in overtly antagonistic interactions; (ii) sanctions are critical for long-term mutualism stability when providing benefits to a host is costly, and (iii) there are general principles that help maintain cooperation both within and among species.

Efficient transformation and regeneration of fig (Ficus carica L.) via somatic embryogenesis.

Fig is one of the most important fruit trees in Egypt. It used to constitute the major source of income for the inhabitants of the western north coast of Egypt. Since 1993 fig cultivations were threatened by a number of factors including virus, insect and mite infections. An efficient system for regeneration and transformation of the common fig Ficus carica L. cultivar Sultani (fresh consumption) was required to conserve fig cultivation in the area. The effect of different combinations of BA and NAA/2,4-D and kinetin on callus formation from leaf segments were studied. Results showed that the best medium for callus formation was MS supplemented with 2.0 mg/l 2,4-D and 0.2 mg/l kinetin. The best plantlet differentiation was obtained at concentrations of 30 mg/l 2iP and 7 mg/l TDZ with 0.25 mg/l NAA (with a regeneration efficiency of 83 and 79%, respectively). On the other hand, the obtained callus failed to induce organogenesis on media containing a combination of BA and kinetin. The highest shoot formation percentage (89%) was obtained when using 2 mg/l TDZ and 4 mg/l 2iP. The highest percentage of shoots forming roots (95%) was obtained when using MS medium supplemented with 1.0 mg/l IBA. Explants were transformed using Agrobacterium and microprojectile bombardment using the plasmid pISV2678 which harbors the gus-intron and bar genes. Results showed that the highest transformation efficiency using the Agrobacterium (17.5%) was obtained when explants were co-cultivated with the bacteria for 30 min. The highest transformation efficiency recorded using the microprojectile bombardment (12%) was obtained with 2.0 µg DNA per shot at 1,100 psi and a distance of 6 cm repeated twice. The transgenic nature of regenerated plants was confirmed by PCR analysis, histochemical GUS assay and leaf painting assay.

Presence of triploid cytotypes in the common fig (Ficus carica L.).

Ficus carica (2n = 26) is one of the oldest fruit trees of the Mediterranean basin. Recently there has been increasing interest in this species, in particular for questions related to germplasm such as genetic diversity and cultivar identification. This study was undertaken to gain more knowledge of F. carica cytogenetics and provide data useful for the characterization of its germplasm. Karyomorphological analysis and physical mapping of 18S-25S and 5S rRNA genes by the FISH technique contributed to defining the basic traits of the chromosome complement of F. carica. However, the most interesting result was the discovery of triploid (2n = 39) cytotypes of the cultivated common fig. This result demonstrates the importance of cytogenetic investigations in studies of fig germplasm and emphasizes the role of cross-fertilization as a source of variability not only in wild populations but also in cultivated forms. The results of pollen analysis suggest spontaneous sexual polyploidization as a possible origin of triploid cytotypes. Further studies are necessary to clarify the origin and effective spreading of polyploidy, the presence of other ploidy levels, and their distribution in wild and cultivated forms.

Characterization of oil bodies in jelly fig achenes.

Thin-layer chromatography analysis revealed that the contents stored in oil bodies isolated from jelly fig (Ficus awkeotsang Makino) achenes were mainly neutral lipids (>90% triacylglycerols and approximately 5% diacylglycerols). Fatty acids released from the neutral lipids of achene oil bodies were highly unsaturated (62.65% alpha-linolenic acid, 18.24% linoleic acid, and 10.62% oleic acid). The integrity of isolated oil bodies was presumably maintained via electronegative repulsion and steric hindrance provided by their surface proteins. Immunological cross-recognition using antibodies against sesame oil-body proteins indicated that two oleosin isoforms and one caleosin were present in these oil bodies. MALDI-MS analyses confirmed that the three full-length cDNA fragments obtained by PCR cloning from maturing achenes encoded the two jelly fig oleosin isoforms and one caleosin identified by immunological screening.

Interspecific variation of plant traits associated with resistance to herbivory among four species of Ficus (moraceae).

BACKGROUND AND AIMS: To understand the defensive characteristics of interspecies varieties and their responses to herbivory damage, four species of Ficus plants (Ficus altissima, F. auriculata, F. racemosa and F. hispida) were studied. They were similar in life form, but differed in successional stages. Of these, Ficus altissima is a late successional species, F. hispida is a typical pioneer and F. auriculata and F. racemosa are intermediate successional species. We addressed the following questions: (1) What is the difference in plant traits among the four species and are these traits associated with differences in herbivory damage levels? (2) What is the difference in the damage-induced changes among the four species? METHODS: Herbivory damage was measured in the field on randomly planted seedlings of the four species of the same age. Defences to herbivory were also tested by feeding leaves of the four species to larvae of Asota caricae in the laboratory. A total of 14 characters such as water content, thickness, toughness, pubescence density on both sides, leaf expansion time, lifetime and the contents of total carbon (C), nitrogen (N), phosphorous (P), potassium (K), magnesium (Mg) and calcium (Ca) were measured. Leaf calcium oxalate crystal (COC) density, total Ca and N content, leaf toughness and height were measured to investigate induced responses to artificial herbivory among the four species. KEY RESULTS: and conclusions Herbivory damage in the four studied species varied greatly. The pioneer species, F. hispida, suffered the most severe herbivory damage, while the late successional species, F. altissima, showed the least damage. A combination of several characteristics such as high in content of N, Ca and P and low in leaf toughness, lifetime and C : N ratio were associated with increased herbivore damage. The late successional species, F. altissima, might also incorporate induced defence strategies by means of an increase in leaf COC and toughness.

Speciation in fig pollinators and parasites.

Here we draw on phylogenies of figs and fig wasps to suggest how modes of speciation may be affected by interspecific interactions. Mutualists appear to have cospeciated with their hosts to a greater extent than parasites, which showed evidence of host shifting. However, we also repeatedly encountered a pattern not explained by either cospeciation or host switching. Sister species of fig parasites often attack the same host in sympatry, and differences in ovipositor length suggest that parasite speciation could result from divergence in the timing of oviposition with respect to fig development. These observations on fig parasites are consistent with a neglected model of sympatric speciation.