Development and characterization of microsatellite markers for two dioecious Ficus species

Microsatellite markers for Ficus montana and Ficus septica were developed using genomic libraries enriched for di‐, tri‐ and tetranucleotide repeats. The subsets of five and three best scorable primer pairs were characterized on 24 F. montana and 36 F. septica individuals, respectively. For F. montana, loci showed five to 14 alleles per locus and expected heterozygosities ranged between 0.23 and 0.87. For F. septica, loci showed three to five alleles per locus and expected heterozygosities ranged between 0.36 and 0.49. Four primer pairs (two from each subset) cross‐amplified in the other species, indicating transportability of the markers within the genus Ficus.     

Characterization of microsatellite loci in the African fig Ficus sycomorus L. (Moraceae)

Microsatellite loci were characterized in the African fig tree Ficus sycomorus in order to investigate patterns of pollination and gene flow in this species. The loci characterized included new loci isolated from F. sycomorus and a single locus originally developed in Ficus carica. In total 12 loci were polymorphic when tested in between eight and 79 Namibian F. sycomorus individuals. Three of the new F. sycomorus loci were found to be polymorphic in cultivars of the edible fig F. carica suggesting a selection of these loci will be useful for population studies in other fig species.     

Identification and characterization of microsatellite loci in the common fig (Ficus carica L.) and representative species of the genus Ficus

We developed microsatellites in fig (Ficus carica L.). A TC and TG‐enriched genomic library was screened, and after sequencing, primers were designed for 20 microsatellites. Eight primer pairs produced amplification products that were both interpretable and polymorphic in 14 fig cultivars and two French wild‐growing populations of F. carica (n₁ = 9 and n₂ = 10). Number of alleles per locus ranged from three to six. Except for one microsatellite locus, the observed heterozygosity was higher than the expected value. The F. carica microsatellites gave amplification products in 17 other Ficus species in 86% of the cases.     

Development and characterization of microsatellite markers for a fig‐pollinating wasp (Ceratosolen sp. 1) of Ficus septica

For an undescribed fig‐pollinating wasp (Ceratosolen sp. 1) of Ficus septica, we isolated 15 polymorphic microsatellite markers from a genomic library partially enriched for GATA and CAG repeats. Polymorphism of these loci was evaluated by genotyping 40 female individuals collected from different fruits of host trees in southern Taiwan. The number of alleles per locus ranged from three to 15 and the observed heterozygosity of each locus ranged from 0.100 to 0.775. These loci would be suitable for further studies concerning population genetics, hybridization and sex ratio adjustment of the coexisting fig‐pollinating wasps.     

Phenology of Ficus racemosa in Xishuangbanna,Southwest China1

Leaf and fig phenology (including leafing, flowering, and fruiting) and syconium growth of Ficus racemosa were studied in Xishuangbanna, China. Leaffall and flushing of F. racemosa occurred twice yearly: in mid‐dry season (December to March) and mid‐rainy season (July to September). The adult leaf stage of the first leaf production was remarkably longer than that of the second. F. racemosa bears syconia throughout the year, producing 4.76 crops annually. Asynchronous fig production was observed at a population level. Fig production was independent of leafing. Fig production peaks were not evident, but fluctuation was clear. Diameter growth rates of syconium were normally higher in early developmental stages than in later stages, and reached a peak coinciding with the female flower phase. The mean ± SD of syconium diameter of the female flower phase was 2.19 ± 0.36 cm, and reached 3.67 ± 0.73 cm of the male flower phase. Syconium diameter and receptacle cavity quickly enlarged at the female and male flower phases. Monthly diameter increment of the syconium was primarily affected by average monthly temperature, rather than rainfall or relative humidity.     

Differential responses of photosystems I and II to seasonal drought in two Ficus species

Hemiepiphytic Ficus species exhibit more conservative water use strategy and are more drought-tolerant compared with their non-hemiepiphytic congeners, but a difference in the response of photosystem I (PSI) and photosystem II (PSII) to drought stress has not been documented to date. The enhancement of non-photochemical quenching (NPQ) and cyclic electron flow (CEF) have been identified as important mechanisms that protect the photosystems under drought conditions. Using the hemiepiphytic Ficus tinctoria and the non-hemiepiphytic Ficus racemosa, we studied the water status and the electron fluxes through PSI and PSII under seasonal water stress. Our results clearly indicated that the decline in the leaf predawn water potential (ψ_pd), the maximum photosynthetic rate (A_max) and the predawn maximum quantum yield of PSII (F_v/F_m) were more pronounced in F. racemosa than in F. tinctoria at peak drought. The F_v/F_m of F. racemosa was reduced to 0.69, indicating net photoinhibition of PSII. Concomitantly, the maximal photo-oxidizable P700 (P_m) decreased significantly in F. racemosa but remained stable in F. tinctoria. The fraction of non-photochemical quenching [Y(NPQ)] and the ratio of effective quantum yield of PSI to PSII [Y(I)/Y(II)] increased for both Ficus species at peak drought, with a stronger increase in F. racemosa. These results indicated that the enhancement of NPQ and the activation of CEF contributed to the photoprotection of PSI and PSII for both Ficus species under seasonal drought, particularly for F. racemosa.     

Development and characterization of microsatellite markers for a monoecious Ficus species,Ficus insipida,and cross‐species amplification among different sections of Ficus

The animal‐mediated pollination and seed‐dispersal mutualisms of Ficus species give them key roles in tropical ecosystems, but may make them vulnerable to habitat fragmentation. The development of highly polymorphic markers is needed to analyse their genetic diversity and investigate the effects of fragmentation on gene flow. Of thirteen microsatellite loci isolated from Ficus insipida, a monoecious species in French Guiana, eleven were polymorphic (two to six alleles each). High levels of variation were found among loci; expected heterozygosities ranged from 0.151 to 0.715. All markers revealed a broad cross‐species affinity when tested in 23 other Ficus species.     

Ficus racemosa is pollinated by a single population of a single agaonid wasp species in continental South‐East Asia

High specificity in the Ficus‐agaonid wasp mutualism has lead to the assumption of a mostly ‘one‐to‐one’ relationship, albeit with some exceptions. This view has been challenged by new molecular data in recent years, but surprisingly little is known about local and spatial genetic structuring of agaonid wasp populations. Using microsatellite markers, we analysed genetic structuring of Ceratosolen fusciceps, the fig wasp pollinating Ficus racemosa, a fig tree species widely distributed from India to Australia. In sampling stretching from the south of China to the south of Thailand we found evidence for only a single pollinating wasp species in continental South‐East Asian mainland. We found no evidence for the co‐occurrence of cryptic species within our subcontinent sampling zone. We observed no spatial genetic structure within sites and only limited structuring over the whole sampling zone, suggesting that F. racemosa is pollinated by a single population of a single agaonid wasp species all over continental South‐East Asia. An additional sample of wasps collected on F. racemosa in Australia showed clear‐cut genetic differentiation from the Asian continent, suggesting allopatric divergence into subspecies or species. We propose that the frequent local co‐occurrence of sister species found in the literature mainly stems from contact zones between biogeographic regions, and that a single pollinator species over wide areas might be the more common situation everywhere else.     

Host associations of the strangler fig Ficus watkinsiana in a subtropical Queensland rain forest

Abstract The distribution of epiphytic organisms is limited by the availability of, and dispersal to suitable hosts. We examined the distribution of a hemi‐epiphytic strangler fig, Ficus watkinsiana (Moraceae) in Cooloola National Park (Queensland, Australia), in order to determine whether this species exhibits a preference for certain host species and why. We assessed host bark roughness and flakiness, fruit type, and size to explain the observed distribution of F. watkinsiana. We surveyed over 1900 potential host trees of the 30 most common forest canopy species and found that host size measured by diameter at breast height accounted for most variation in fig prevalence (Binary Logistic Regression log‐likelihood = ?588.178, G = 314.494, d.f. = 1, P < 0.005). After controlling for host size, F. watkinsiana prevalence still differed significantly between host species (χ² = 54.612, d.f. = 24, P < 0.005), a difference that was only partly explained by variation in the bark roughness of host trunks. These results suggest that variation in the rate at which tree species host strangler figs are primarily related to individual tree size – figs may simply be more likely to colonize and thrive upon host species that grow larger.     

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

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

Isolation of microsatellite loci in the pollinating fig wasp of Ficus hispida, Ceratosolen solmsi

Microsatellite loci were isolated for Ceratosolen solmsi , pollinator of the dioecious Ficus hispida. We developed nine polymorphic microsatellite loci based on the method of polymerase chain reaction isolation of microsatellite arrays (PIMA). Enrichment of genomic libraries was performed by random amplified polymorphic DNA (RAPD). A subset of 38 positive clones was sequenced; 15 clones showed microsatellite loci. We tested 15 designed primer pairs and nine of them produced polymorphic amplification in 48 individual wasps collected from different fruits of the dioecious host fig Ficus hispida in China. Among the 48 individuals, 49 alleles were obtained at the nine loci. The observed heterozygosity ranged between 0.357 and 0.634.     

Fine‐scale Population Genetic Structure of Two Dioecious Indian Keystone Species,Ficus hispida and Ficus exasperata (Moraceae)

Although Ficus (Moraceae) is a keystone plant genus in the tropics, providing resources to many frugivorous vertebrates, its population genetic structure, which is an important determinant of its long‐term survival, has rarely been investigated. We examined the population genetic structure of two dioecious fig species (Ficus hispida and Ficus exasperata) in the Indian Western Ghats using co‐dominant nuclear microsatellite markers. We found high levels of microsatellite genetic diversity in both species. The regression slopes between genetic relationship coefficients (f_ij) and spatial distances were significantly negative in both species indicating that, on average, individuals in close spatial proximity were more likely to be related than individuals further apart. Mean parent–offspring distance (σ) calculated using these slopes was about 200 m in both species. This should be contrasted with the very long pollen dispersal distances documented for monoecious Ficus species. Nevertheless, overall population genetic diversity remained large suggesting immigrant gene flow. Further studies will be required to analyze broader scale patterns.     

Drastic post‐LGM expansion and lack of historical genetic structure of a subtropical fig‐pollinating wasp (Ceratosolen sp. 1) of Ficus septica in Taiwan

The climatic oscillations of the last glacial period have had profound influences on the demography and levels of genetic diversity of extant species. Molecular evidence of glacial effects on temperate species has been well documented, whereas little is known regarding that on subtropical species. Here we present analyses based on partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene (1052 bp) and genotypes at 15 microsatellite loci to investigate the historical demography, population structure and ongoing gene flow of an undescribed fig‐pollinating wasp (Ceratosolen sp. 1) of Ficus septica in subtropical Taiwan. Reconstructed historical demography based on the coalescent tree of COI sequences suggests that C. sp. 1 has undergone a drastic population expansion which was tightly coupled with climatic changes since the last glacial maximum (LGM). The magnitude of the population size change was approximately 500‐fold, indicating that the population of this wasp and its host was likely highly compressed during the last glacial period. The lack of significant population differentiation (F_ST < 0.02, for all pairwise F_ST values) may be due to rapid postglacial expansion facilitated by long‐distance dispersal, although a low frequency of first‐generation migrants was detected. Our results clearly demonstrate how recent climatic changes since the LGM and dispersal ability have jointly shaped the genetic composition of a subtropical fig‐pollinating wasp.     

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 2850 km, 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.     

Non‐pollinating cheater wasps benefit from seasonally poor performance of the mutualistic pollinating wasps at the northern limit of the range of Ficus microcarpa

1. Species interactions in tightly bound ecological mutualisms often feature highly specialised species' roles in which competitive exclusion may preclude multi‐species coexistence. Among the 800 fig (Ficus) species, it was originally considered that each was pollinated by their own wasp (Agaonidae). However, recent investigations show that this ‘one‐to‐one’ rule often breaks down, as fig species regularly host multiple agaonids but in ways suggesting that competitive processes still mediate biodiversity outcomes. 2. A phenological survey was conducted of the fig–fig wasp pair, Ficus microcarpa and its associated pollinating wasp, alongside its sister species, the cheating wasp, in Xishuangbanna, China. 3. Reproductive output underwent extreme seasonal variation. Seed and pollinator production fell markedly during cooler, drier months, although high levels of fig production continued. However, this resource was predominantly utilised by the cheater species, which offers no pollination services. Pollinators and cheaters rarely co‐occur, suggesting that temporal coexistence is constrained by competition for access to figs. 4. The overall findings indicate periodic rearrangements of mutualism dynamics, probably resulting from a strongly seasonal environment. Sympatric co‐occurrence may result from a window of opportunity for a functionally divergent agaonid, potentially due to constraints on the main pollinator in adapting to variable year‐round conditions that prevent competitive exclusion.     

Development of microsatellite markers in the common fig,Ficus carica L.

We present a new set of 15 polymorphic microsatellite primer sequences developed from Ficus carica L. The variability of specific microsatellite regions was assessed in wild population of figs from the northern Adriatic coast and all 15 primer pairs showed single‐locus amplification with a total of 65 alleles and an observed heterozygosity ranging from 0.285 to 0.863. The 15 new microsatellite loci represent a significant tool for population genetic structure studies and will be further used to investigate the origin and maintenance of genetic variation within and between populations of figs along the Adriatic coastal region.     

One step ahead: a parasitoid disperses farther and forms a wider geographic population than its fig wasp host

The structure of populations across landscapes influences the dynamics of their interactions with other species. Understanding the geographic structure of populations can thus shed light on the potential for interacting species to co‐evolve. Host–parasitoid interactions are widespread in nature and also represent a significant force in the evolution of plant–insect interactions. However, there have been few comparisons of population structure between an insect host and its parasitoid. We used microsatellite markers to analyse the population genetic structure of Pleistodontes imperialis sp. 1, a fig‐pollinating wasp of Port Jackson fig (Ficus rubiginosa), and its main parasitoid, Sycoscapter sp. A, in eastern Australia. Besides exploring this host–parasitoid system, our study also constitutes, to our knowledge, the first study of population structure in a nonpollinating fig wasp species. We collected matched samples of pollinators and parasitoids at several sites in two regions separated by up to 2000 km. We found that pollinators occupying the two regions represent distinct populations, but, in contrast, parasitoids formed a single population across the wide geographic range sampled. We observed genetic isolation by distance for each species, but found consistently lower F_ST and R_ST values between sites for parasitoids compared with pollinators. Previous studies have indicated that pollinators of monoecious figs can disperse over very long distances, and we provide the first genetic evidence that their parasitoids may disperse as far, if not farther. The contrasting geographic population structures of host and parasitoid highlight the potential for geographic mosaics in this important symbiotic system.     

Holocene southward expansion in seasonally dry tropical forests in South America: phylogeography of Ficus bonijesulapensis (Moraceae)

We conducted a phylogeographical and niche modelling study of the tree Ficus bonijesulapensis, endemic to Brazilian seasonally dry tropical forests (SDTFs), in order to evaluate the effects of Quaternary climatic fluctuations on population dynamics. The trnQ–5′rps16 region of plastid DNA was sequenced from 15 populations. Three phylogeographical groups were identified by the median‐joining algorithm network and spatial analysis of molecular variance (SAMOVA) (F_CT = 0.591): a central‐west, a central‐east and a scattered group. The central groups had higher total haplotype and nucleotide diversities than the scattered group. Ecological niche modelling suggested that, since the Last Interglacial (130 kyr bp ), the central and north regions have been relatively stable, whereas the southern region of the species distribution has been less stable. The phylogeographical groups showed concordance with the floristic units described for SDTFs. The low genetic diversity, unimodal mismatch distribution and unfavourable climatic conditions in the southern region suggest a recent southward expansion of the range of the species during the Holocene, supporting the hypothesis of the southward expansion of SDTFs during this period. The central and northern regions of the current distribution of F. bonijesulapensis, which are consistent with arboreal caatinga and rock outcrop floristic units, were potential refugia during Quaternary climatic fluctuations. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 189–201.     

Occurrence of crown gall disease on Ficus benjamina in Fars and Isfahan provinces of Iran

Ficus benjamina, commonly known as weeping fig, Benjamin’s fig or Ficus tree is a species of flowering plant in the family of Moraceae. It is native to south and south-east Asia and Australia. Crown gall tumours were collected from branches of one-year-old weeping fig (F. benjamina L.) trees. A total of 50 strains of Agrobacterium tumefaciens were isolated from diseased Ficus plants and their morphological, molecular and biochemical characteristics were studied; pathogenicity tests on tomato, F. benjamina and Bryophyllum daigremontianum were also conducted. Based on the biochemical characteristics, pathogenicity test and PCR amplification of 730?bp fragment using VCR\VCF primers, the tested bacterial strains were identified as A. tumefaciens. This is the first report of crown gall on F. benjamina in Isfahan and Fars provinces of Iran.     

Genetic and physiological data suggest demographic and adaptive responses in complex interactions between populations of figs (Ficus pumila) and their pollinating wasps (Wiebesia pumilae)

To study interactions between host figs and their pollinating wasps and the influence of climatic change on their genetic structures, we sequenced cytoplasmic and nuclear genes and genotyped nuclear microsatellite loci from two varieties of Ficus pumila, the widespread creeping fig and endemic jelly fig, and from their pollinating wasps, Wiebesia pumilae, found in Taiwan and on nearby offshore islands. Great divergence in the mitochondrial cytochrome c oxidase subunit I (mtCOI) with no genetic admixture in nuclear markers indicated that creeping‐ and jelly‐fig wasps are genetically distinct. Compared with creeping‐fig wasps, jelly‐fig wasps also showed better resistance under cold (20 °C) than warm (25 and 30 °C) conditions in a survival test, indicating their adaptation to a cold environment, which may have facilitated population expansion during the ice age as shown by a nuclear intron and 10 microsatellite loci. An excess of amino acid divergence and a pattern of too many rare mtCOI variants of jelly‐fig wasps as revealed by computer simulations and neutrality tests implied the effect of positive selection, which we hypothesize was associated with the cold‐adaptation process. Chloroplast DNA of the two fig plants was completely segregated, with signs of genetic admixture in nuclear markers. As creeping‐ and jelly‐fig wasps can pollinate creeping figs, occasional gene flow between the two figs is thus possible. Therefore, it is suggested that pollinating wasps may be playing an active role in driving introgression between different types of host fig.