Genomes of 12 fig wasps provide insights into the adaptation of pollinators to fig syconia.

Figs and fig pollinators are one of the few classic textbook examples of obligate pollination mutualism. The specific dependence of fig pollinators on the relatively safe living environment with sufficient food sources in the enclosed fig syconia implies that they are vulnerable to habitat changes. However, there is still no extensive genomic evidence to reveal the evolutionary footprint of this long-term mutually beneficial symbiosis in fig pollinators. In fig syconia, there are also non-pollinator species. The non-pollinator species differ in their evolutionary and life histories from pollinators. We conducted comparative analyses on 11 newly sequenced fig wasp genomes and one previously published genome. The pollinators colonized the figs approximately 66.9 million years ago, consistent with the origin of host figs. Compared with non-pollinators, many more genes in pollinators were subject to relaxed selection. Seven genes were absent in pollinators in response to environmental stress and immune activation. Pollinators had more streamlined gene repertoires in the innate immune system, chemosensory toolbox, and detoxification system. Our results provide genomic evidence for the differentiation between pollinators and nonpollinators. The data suggest that owing to the long-term adaptation to the fig, some genes related to functions no longer required are absent in pollinators.

Evaluation of endogenous reference genes in Bactrocera cucurbitae by qPCR under different conditions.

Bactrocera cucurbitae (melon flies) are prominent invasive pests in southern China. To screen for a stable reference gene in melon flies suitable for comparing tissue samples subjected to different conditions in four categories (temperature, insect stage, days of age and gender), the expression of 12 candidate reference genes under different treatment conditions was analyzed by real-time fluorescent quantitative PCR. The results obtained from a comprehensive analysis with geNorm, NormFinder, BestKeeper and RefFinder software showed that the most stable reference gene was RPL60, and the least stable reference gene was actin-5. We used a heat shock protein gene (HSP-90) to verify the results, and the conclusion was consistent. When the reference gene RPL60 was used as the reference gene, the relative expression of HSP-90 was essentially constant with the prolongation of treatment time. When actin-5 was used, HSP-90 expression changed markedly with treatment time. The results of this study can be used for further research on gene expression inBactrocera cucurbitae.

Evaluation of chromatic cues for trapping Bactrocera tau.

BACKGROUND: Trapping technology based on chromatic cues is an important strategy in controlling Tephritidae (fruit flies). The objectives of this present study were to evaluate the preference of Bactrocera tau for different chromatic cues, and to explore an easy method to print and reproduce coloured paper. RESULTS: Chromatic cues significantly affected the preference of adult B. tau. Wavelengths in the 515-604 nm range were the suitable wavelengths for trapping B. tau. Different-day-old B. tau had different colour preferences. Virtual wavelengths of 595 nm (yellow) and 568 nm (yellowish green) were the optimum wavelengths for trapping 5-7-day-old B. tau and 30-32-day-old B. tau respectively. The trap type and height significantly influenced B. tau attraction efficiency. The number of B. tau on coloured traps hung perpendicular to plant rows was not significantly higher than the number on traps hung parallel to plant rows. CONCLUSION: The quantisation of colour on the basis of Bruton's wavelength to RGB function can serve as an alternative method for printing and reproducing coloured paper, but a corrected equation should be established between the theoretical wavelength and actual wavelength of coloured paper. Results show that a compound paper coloured yellow (595 nm) and yellowish green (568 nm) installed at 60 and 90 cm above the ground shows the maximum effect for trapping B. tau. © 2016 Society of Chemical Industry.

Large proportion of genes in one cryptic WO prophage genome are actively and sex-specifically transcribed in a fig wasp species.

BACKGROUND: Cryptic prophages are genetically defective in their induction and propagation, and are simply regarded as genetic remnants. There are several putative cryptic WO prophages in the sequenced Wolbachia genomes. Whether they are lytic is unclear and their functions are poorly understood. Only three open reading frames (ORFs) in cryptic WO prophages have been reported to be actively transcribed. RESULTS: In this study, we comprehensively examined the transcription of the only cryptic WO prophage (WOSol) in a Wolbachia strain that infects a fig wasp, Ceratosolen solmsi (Agaonidae, Chalcidoidea). By analyzing the transcriptions of all the ORFs of WOSol in both sexes of C. solmsi, using qualitative and quantitative methods, we demonstrated that i) a high percentage of ORFs are actively transcribed (59%, 17/29); ii) the expression of these ORFs is highly sex-specific, with a strong male bias (three in females and 15 in males); iii) an ank (ankyrin-domain-containing) gene actively transcribed in both wasp sexes is more highly expressed in males. CONCLUSIONS: A large proportion of the genes in the cryptic WO prophage WOSol are expressed, which overturns the concept that cryptic prophages are simply genetically defective. The highly sex-specific expression patterns of these genes in the host suggest that they play important roles in Wolbachia biology and its reproductive manipulation of its insect host, particularly through the males.

[Combination of narrow band imaging (NBI) and autofluorescence bronchoscopy (AFB) in the assessment of central lung cancer].

OBJECTIVE: To assess the diagnostic value of narrow-band imaging(NBI) in the diagnosis of central lung cancer. METHODS: Patients (n = 153) suspected of having lung cancer underwent white light bronchoscopy(WLB), NBI and autofluorescence bronchoscopy(AFB) in turn. At least 3 biopsies in each case were taken from sites visualized as lesions. The sensitivity and specificity of NBI, AFB and combination of NBI and AFB were compared. RESULTS: There were 106 male (69.3%) and 47 female patients (30.7%). By NBI, 91 and 62 cases were positive and negative respectively. The sensitivity and specificity of NBI were 63.5% (87/137) and 75.0% (12/16) respectively. By AFB, 140 and 13 cases were positive and negative respectively. The sensitivity and specificity of AFB were 94.2% (129/137) and 87.5% (5/16) respectively. By NBI combined with AFB, 133 and 20 cases were positive and negative respectively, the sensitivity and specificity being 95.6% (131/137) and 87.5% (14/16) respectively. The difference of specificity between NBI plus AFB and AFB alone was significant (P < 0.01), but the difference of sensitivity between NBI plus AFB and AFB alone(P > 0.05) was not. The difference of specificity between NBI plus AFB and NBI alone was significant (P < 0.01), but the P value of specificity between NBI plus AFB and NBI was 0.03. CONCLUSION: Combination of NBI and AFB could increase the specificity of lung cancer diagnosis compared to AFB alone.

Evidence for the circadian gene period as a proximate mechanism of protandry in a pollinating fig wasp.

Protandry in insects is the tendency for adult males to emerge before females and usually results from intra-sexual selection. However, the genetic basis of this common phenomenon is poorly understood. Pollinating fig wasp (Agaonidae) larvae develop in galled flowers within the enclosed inflorescences ('figs') of fig trees. Upon emergence, males locate and mate with the still galled females. After mating, males release females from their galls to enable dispersal. Females cannot exit galls or disperse from a fig without male assistance. We sampled male and female Ceratosolen solmsi (the pollinator of Ficus hispida) every 3 h over a 24 h emergence period, and then measured the expression of five circadian genes: period (per), clock (clk), cycle (cyc), pigment-dispersing factor (pdf) and clockwork orange (cwo). We found significant male-biased sexual dimorphism in the expression of all five genes. per showed the greatest divergence between the sexes and was the only gene rhythmically expressed. Expression of per correlated closely with emergence rates at specific time intervals in both male and female wasps. We suggest that this rhythmical expression of per may be a proximate mechanism of protandry in this species.

Adaptive evolution of the circadian gene timeout in insects.

Most insects harbor two paralogous circadian genes, namely timeout and timeless. However, in the Hymenoptera only timeout is present. It remains unclear whether both genes, especially timeout in hymenopteran insects, have distinct evolutionary patterns. In this study, we examine the molecular evolution of both genes in 25 arthropod species, for which whole genome data are available, with addition of the daily expression of the timeout gene in a pollinating fig wasp, Ceratosolen solmsi (Hymenoptera: Chalcidoidea: Agaonidae). Timeless is under stronger purifying selection than timeout, and timeout has positively selected sites in insects, especially in the Hymenoptera. Within the Hymenoptera, the function of timeout may be conserved in bees and ants, but still evolving rapidly in some wasps such as the chalcids. In fig wasps, timeout is rhythmically expressed only in females when outside of the fig syconium but arrhythmically in male and female wasps inside the syconium. These plastic gene expressions reflect adaptive differences of males and females to their environment.

Delimitation and description of the immature stages of a pollinating fig wasp, Ceratosolen solmsi marchali Mayr (Hymenoptera: Agaonidae).

The mutualism between fig trees and their wasp pollinators is a model system for many ecological and evolutionary studies. However, the immature stages of pollinating fig wasps have rarely been studied. We monitored developing fig wasps of known ages and performed a series of dissections at 24 h intervals to identify key developmental traits of Ceratosolen solmsi marchali Mayr (Hymenoptera: Agaonidae), a pollinator of Ficus hispida L. (Moraceae). We identified where in the Ficus ovary eggs were deposited and time to hatch. We were also able to identify the timing and key underlying characters of five larval instars, three sub-pupal stages, and a single prepupal stage. We provide detailed morphological descriptions for the key stages and report some behavioral observations of the wasps in the several developmental stages we recorded. Scanning electron microscope images were taken.

Obligate mutualism within a host drives the extreme specialization of a fig wasp genome.

BACKGROUND: Fig pollinating wasps form obligate symbioses with their fig hosts. This mutualism arose approximately 75 million years ago. Unlike many other intimate symbioses, which involve vertical transmission of symbionts to host offspring, female fig wasps fly great distances to transfer horizontally between hosts. In contrast, male wasps are wingless and cannot disperse. Symbionts that keep intimate contact with their hosts often show genome reduction, but it is not clear if the wide dispersal of female fig wasps will counteract this general tendency. We sequenced the genome of the fig wasp Ceratosolen solmsi to address this question. RESULTS: The genome size of the fig wasp C. solmsi is typical of insects, but has undergone dramatic reductions of gene families involved in environmental sensing and detoxification. The streamlined chemosensory ability reflects the overwhelming importance of females finding trees of their only host species, Ficus hispida, during their fleeting adult lives. Despite long-distance dispersal, little need exists for detoxification or environmental protection because fig wasps spend nearly all of their lives inside a largely benign host. Analyses of transcriptomes in females and males at four key life stages reveal that the extreme anatomical sexual dimorphism of fig wasps may result from a strong bias in sex-differential gene expression. CONCLUSIONS: Our comparison of the C. solmsi genome with other insects provides new insights into the evolution of obligate mutualism. The draft genome of the fig wasp, and transcriptomic comparisons between both sexes at four different life stages, provide insights into the molecular basis for the extreme anatomical sexual dimorphism of this species.

Host sex-specific parasites in a functionally dioecious fig: a preference way of adaptation to their hosts.

Host-parasites interaction is a common phenomenon in nature. Diffusive coevolution might maintain stable cooperation in a fig-fig wasps system, in which the exploiter might diversify their genotype, phenotype, or behavior as a result of competition with pollinator, whereas the figs change flower syconia, fruits thickness, and syconia structure. In functionally dioecious Ficus auriculata, male figs and female figs contain two types of florets on separate plant, and share high similarities in outside morphology. Apocryptophagus (Sycophaginae, Chalcidoidea, Hymenoptera) is one of few groups of nonpollinating fig wasps that can reproduce within both male and female figs. On the basis of the morphology and DNA barcoding, evidence from partial sequences of mitochondrial cytochrome c oxidase I and nuclear internal transcribed spacer 2, we found that there are two nonsibling Apocryptophagus species living on male and female F. auriculata figs, respectively. We estimated that these two species diverged about 19.2 million years ago. Our study suggests that the host shift from Ficus variegate or Ficus prostrata fig species to male figs is a preference way for Apocryptophagus wasps to adapt to the separation of sexual function in diecious figs. Furthermore, to escape the disadvantage or sanction impact of the host, the exploiter Apocryptophagus wasps can preferably adapt to exploiting each sex of the figs, by changing their oviposition, niche shift, and habitat.

Pollinating fig wasp Ceratosolen solmsi adjusts the offspring sex ratio to other foundresses.

Local mate competition theory predicts that offspring sex ratio in pollinating fig wasps is female-biased when there is only one foundress, and increased foundress density results in increased offspring sex ratio. Information of other foundresses and clutch size have been suggested to be the main proximate explanations for sex ratio adjustment under local mate competition. Our focus was to show the mechanism of sex ratio adjustment in a pollinating fig wasp, Ceratosolen solmsi Mayr, an obligate pollinator of the functionally dioecious fig, Ficus hispida Linn., with controlled experiments in the field. First, we obtained offspring from one pollinator and offspring at different oviposition sequences, and found that offspring sex ratio decreased with clutch size, and pollinators produced most of their male offspring at the start of bouts, followed by mostly females. Second, we found that offspring sex ratio increased with foundress density, and pollinators did adjust their offspring sex ratio to other females in the oviposition patches. We suggest that when oviposition sites are not limited, pollinators will mainly adjust their offspring sex ratio to other foundresses independent of clutch size changes, whereas adjusting clutch size may be used to adjust sex ratio when oviposition sites are limited.

Evolution and expression plasticity of opsin genes in a fig pollinator, Ceratosolen solmsi.

Figs and fig pollinators have co-evolved species-specific systems of mutualism. So far, it was unknown how visual opsin genes of pollinators have evolved in the light conditions inside their host figs. We cloned intact full-length mRNA sequences of four opsin genes from a species of fig pollinator, Ceratosolen solmsi, and tested for selective pressure and expressional plasticity of these genes. Molecular evolutionary analysis indicated that the four opsin genes evolved under different selective constraints. Subsets of codons in the two long wavelength sensitive opsin (LW1, LW2) genes were positively selected in ancestral fig pollinators. The ultraviolet sensitive opsin (UV) gene was under strong purifying selection, whereas a relaxation of selective constrains occurred on several amino acids in the blue opsin. RT-qPCR analysis suggested that female and male fig pollinators had different expression patterns possibly due to their distinct lifestyles and different responses to light within the syconia. Co-evolutionary history with figs might have influenced the evolution and expression plasticity of opsin genes in fig pollinators.

Antennal sensilla of female Encarsia guadeloupae Viggiani (Hymenoptera: Aphelinidae), a nymphal parasitoid of the spiraling whitefly Aleurodicus dispersus (Hemiptera: Aleyrodidae).

Encarsia guadeloupae Viggiani (Hymenoptera: Aphelinidae) is a minute, obligate endoparasitoid against the spiraling whitefly Aleurodicus dispersus nymph. The external morphology and distribution of the antennal sensilla of female E. guadeloupae were observed by scanning electron microscopy. Antennae of female E. guadeloupae were geniculate in shape, which consist of scape with a radicula, pedicel, and flagellum. Eight morphological sensilla types were recorded in the females: nonporous sensilla chaetica (CH-NP) and nonporous sensilla trichodea (ST-NP); uniporous sensilla chaetica (CH-UP) and uniporous sensilla trichodea (ST-UP) with a tip pore, basiconic capitate peg sensilla with numerous pores open at the bottom of the grooves; multiporous sensilla placoid (MSP) with the multiporous cuticular structure; uniporous rod-like sensilla (RO-UP) with robust grooved surfaces and the tremendous apical hole; nonporous finger-like sensilla (FI-NP) with abundant pimples at the bulgy, mortar-shaped short stalk. In order to further explore the host location mechanisms and courtship behavior of E. guadeloupae, the possible roles of the antennal sensilla of this species were discussed.

Chaos of Wolbachia sequences inside the compact fig syconia of Ficus benjamina (Ficus: moraceae).

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

Effect of polymorphisms in the ß2-adrenergic receptor on the susceptibility and pulmonary function of patients with chronic obstructive pulmonary disease: a meta analysis.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a heterogeneous and complex disease of which the pathogenesis remains largely unknown. Many factors could influence COPD development and progression. One of them is the genetic risk factor. A severe hereditary deficiency of alpha-1 antitrypsin is the best genetic proof. Four single nucleotide polymorphisms (SNPs) of beta2-adrenergic receptor (ß(2)AR) result in single amino acid substitution. Two loci had been extensively studied and found that they could change the function of ß(2)AR. Two SNPs consist of substitutions of glycine for arginine at amino acid position 16, glutamic acid for glutamine at position 27. Many studies proved that polymorphisms at position 16 and 27 altered the lung function of COPD patients or the patient's susceptibility to the development of COPD. However, there was no exclusive conclusion. Therefore, a meta analysis was done to investigate the effect of polymorphisms in the ß2-adrenergic receptor (ADRB2) gene on the risk of COPD and lung function. METHODS: Comprehensive searches of MEDLINE, Embase, Ovid, HighWire, Cochrane Library, and Chinese databases (CBMdisc, VIP, CNKI, and Wanfang data) from January 1980 to September 2011 were performed, using the keywords: COPD OR chronic obstructive pulmonary disease AND adrenoreceptor OR adrenergic receptor AND polymorphism OR mutation OR variation. Case-control research or cross sectional studies in which diagnosis of COPD met the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines; all the studies reported the ADRB2 genotype at position 16 or 27. Outcomes measured were genotype frequency and forced expiratory volume in the first second (FEV(1)%) in both the case and control. RESULTS: Twelve case-control studies and eight cross-sectional studies were included. Compared to the control (n = 1225), neither Gly/Gly (n = 527) nor Arg/Arg (n = 422) homozygotes at position 16 demonstrated increased susceptibility to COPD, with odds ratios (ORs) of 0.95 (95%CI (0.68, 1.31), z = 0.33, P = 0.740) and 0.82 (95%CI (0.52, 1.28), z = 0.88, P = 0.381), respectively. Similar results were obtained for position 27, with ORs of 0.97 (95%CI (0.77, 1.23), z = 0.21, P = 0.833) for Glu/Glu homozygotes (n = 357) and 0.82 (95%CI (0.53, 1.29), z = 0.85, P = 0.393) for Gln/Gln homozygotes (n = 704) (control = 1183). In patients with COPD, Arg/Arg homozygotes (n = 41) had a similar FEV1% compared with Gly/Gly homozygotes (n = 102) (standardized mean difference (SMD) = 0.88, 95%CI (-0.85, 2.62), z = 1.00, P = 0.319). The genotype distribution was different between Caucasian and Asian populations (all P < 0.05 except the genotype Arg/Gly) for both position 16 and 27. CONCLUSIONS: Polymorphisms of ADRB2 at positions 16 and 27 did not change the risk of COPD nor affect lung function or disease severity. The genotype distribution for these polymorphisms was different between Caucasian and Asian populations.

Molecular approaches identify known species, reveal cryptic species and verify host specificity of Chinese Philotrypesis (Hymenoptera: Pteromalidae).

Philotrypesis, a major component of the fig wasp community (Hymenoptera: Pteromalidae), is a model taxon for studying male fighting and mating behaviour. Its extreme sexual dimorphism and male polymorphism render species identification uncertain and in-depth research on its ecology, behaviour and other evolutionary topics challenging. The fig wasps' enclosed habitat within the syconia makes their mating behaviour inaccessible, to the extent of matching conspecific females and males. In this study, we combine morphological and molecular analyses to identify species of Philotrypesis sampled from south China and to associate their extraordinarily dimorphic genders and labile male morphologies. Morphological evaluations of females identify 22 species and 28 male morphs. The mitochondrial cytochrome c oxidase I and nuclear internal transcribed spacer 2 data detect 21 species using females, and 15 species among the males. Most of the males match the species as delimited by females. Both markers reveal cryptic species in P. quadrisetosa on Ficus vasculosa. Most species of wasps live on one species of fig but three species co-occur in two hosts (F. microcarpa and F. benjamina), which indicates host switching.

Molecular approaches to identify cryptic species and polymorphic species within a complex community of fig wasps.

Cryptic and polymorphic species can complicate traditional taxonomic research and both of these concerns are common in fig wasp communities. Species identification is very difficult, despite great effort and the ecological importance of fig wasps. Herein, we try to identify all chalcidoid wasp species hosted by one species of fig, using both morphological and molecular methods. We compare the efficiency of four different DNA regions and find that ITS2 is highly effective for species identification, while mitochondrial COI and Cytb regions appear less reliable, possibly due to the interference signals from either nuclear copies of mtDNA, i.e. NUMTs, or the effects of Wolbachia infections. The analyses suggest that combining multiple markers is the best choice for inferring species identifications as any one marker may be unsuitable in a given case.

[Control effect of parasitic Metaphycus parasaissetiae on host Parasaissetia nigra].

A laboratory test was conducted to study the control effect of parasitic Metaphycus parasaissetiae on its host Parasaissetia nigra. The functional reactions of the parasitism conformed to the Holling Type II Equation, but the parameters of the functional reactions varied with temperature. Taking the ratio of instant attack rate to preying time (a/T(h)) as an evaluation index, the preying efficiency at 30 degrees C was the highest, with a/T(h) being 23.4211. There was a stronger interference effect in the functional reactions of the parasitism within M. parasaissetiae populations. With the increase of the population density, the amounts of parasitism decreased gradually. Hassell Equation (E = QP(-m)) could describe the relationships between the searching efficiency of M. parasaissetiae and its population density much precisely within the range of 21 degrees C - 33 degrees C. The interference increased with temperature within the range of 21 degrees C - 27 degrees C, and the interference coefficient reached the highest (0.6626) at 27 degrees C. When the temperature was raised to 30 and 33 degrees C, the interference coefficient decreased to 0.6161 and 0.5916, respectively. In the prophase of egg-laying, the parasitized P. nigra could be entirely controlled by M. parasaissetiae. However, when a few larvae were crawling out, the control effect was declined to 81.4%.

[Validation of HJ-1 HSI red edge parameters based on spectral reflectance of Hyperion].

To validate the HJ-1A HSI red edge indices, spectral reflectance data from EO-1 Hyperion of close date were used to simulate the band reflectance of HJ-1A HSI. Four red edge indices (red vale position, red edge position, red edge slop and red edge swing) were extracted from both simulated and actual HJ-1A HSI band reflectance. Comparisons of the 4 red edge indices between simulated and actual HJ-1A HSI were made to validate the red edge indices product of HJ-1A HSI. The average correlation coefficient of red edge reflectance between actual and simulated HJ-1A HSI is 0.946 and its standard deviation is 0.011, thus a high consistency could be found. The correlation coefficients of red edge indices between simulated and actual HJ-1A HSI were 0.414, 0.543, 0.808 and 0.802 for red edge position, red vale position, the red edge swing and red edge slop respectively. An obvious regular varying trend was found for these 4 red edge indices along different vegetation cover fraction. The standard deviations of differential images between real and simulated HJ-1A HSI red edge indices are 5.75, 1.86, 5.7 e(-4) and 0.024. The result showed that the red edge indices from HJ-1A HSI is consistent with that from simulated indices from Hyperion; the vegetation variation could be effectively reflected in HJ-1A HSI red edge indices.

COI and ITS2 sequences delimit species, reveal cryptic taxa and host specificity of fig-associated Sycophila (Hymenoptera, Eurytomidae).

Although the genus Sycophila has broad host preferences, some species are specifically associated with figs as nonpollinator wasps. Because of their sexual dimorphism, morphological plasticity, cryptic mating behaviour and poorly known biology, species identifications are often uncertain. It is particularly difficult to match conspecific females and males. In this study, we employed two molecular markers, mitochondrial COI and nuclear ITS2, to identify Sycophila from six Chinese fig species. Morphological studies revealed 25 female and male morphs, while sequence results for both genes were consistent in supporting the presence of 15 species, of which 13 were host specialists and two used dual hosts. A single species of Sycophila was respectively found on four fig species, but six species were isolated from Ficus benjamina and a same number was reared from Ficus microcarpa. Sequence results revealed three male morphs in one species and detected two species that were overlooked by morphological analysis.