Male Flower Buds Research Articles

Transcriptome Remodeling in Arabidopsis: A Response to Heterologous Poplar MSL-lncRNAs Overexpression.

Stamens are vital reproductive organs in angiosperms, essential for plant growth, reproduction, and development. The genetic regulation and molecular mechanisms underlying stamen development are, however, complex and varied among different plant species. MSL-lncRNAs, a gene specific to the Y chromosome of Populus deltoides, is predominantly expressed in male flower buds. Heterologous expression of MSL-lncRNAs in Arabidopsis thaliana resulted in an increase in both stamen and anther count, without affecting pistil development or seed set. To reveal the molecular regulatory network influenced by MSL-lncRNAs on stamen development, we conducted transcriptome sequencing of flowers from both wild-type and MSL-lncRNAs-overexpressing Arabidopsis. A total of 678 differentially expressed genes were identified between wild-type and transgenic Arabidopsis. Among these, 20 were classified as transcription factors, suggesting a role for these regulatory proteins in stamen development. GO enrichment analysis revealed that the differentially expressed genes were significantly associated with processes such as pollen formation, polysaccharide catabolic processes, and secondary metabolism. KEGG pathway analysis indicated that MSL-lncRNAs might promote stamen development by upregulating genes involved in the phenylpropanoid biosynthesis pathway. The top three upregulated genes, all featuring the DUF295 domain, were found to harbor an F-box motif at their N-termini, which is implicated in stamen development. Additionally, in transgenic Arabidopsis flowers, genes implicated in tapetum formation and anther development were also observed to be upregulated, implying a potential role for MSL-lncRNAs in modulating pollen development through the positive regulation of these genes. The findings from this study establish a theoretical framework for elucidating the genetic control exerted by MSL-lncRNAs over stamen and pollen development.

Targeted Metabolites and Transcriptome Analysis Uncover the Putative Role of Auxin in Floral Sex Determination in Litchi chinensis Sonn.

Litchi exhibits a large number of flowers, many flowering batches, and an inconsistent ratio of male and female flowers, frequently leading to a low fruit-setting rate. Floral sexual differentiation is a crucial phase in perennial trees to ensure optimal fruit production. However, the mechanism behind floral differentiation remains unclear. The objective of the study was to identify the role of auxin in floral differentiation at the transcriptional level. The results showed that the ratio of female flowers treated with naphthalene acetic acid (NAA) was significantly lower than that of the control stage (M0/F0). The levels of endogenous auxin and auxin metabolites were measured in male and female flowers at different stages of development. It was found that the levels of IAA, IAA-Glu, IAA-Asp, and IAA-Ala were significantly higher in male flowers compared to female flowers. Next-generation sequencing and modeling were employed to perform an in-depth transcriptome analysis on all flower buds in litchi 'Feizixiao' cultivars (Litchi chinensis Sonn.). Plant hormones were found to exert a significant impact on the litchi flowering process and flower proliferation. Specifically, a majority of differentially expressed genes (DEGs) related to the auxin pathway were noticeably increased during male flower bud differentiation. The current findings will enhance our comprehension of the process and control mechanism of litchi floral sexual differentiation. It also offers a theoretical foundation for implementing strategies to regulate flowering and enhance fruit production in litchi cultivation.

Comparative Proteomic Analysis of Floral Buds before and after Opening in Walnut (Juglans regia L.).

The walnut (Juglans regia L.) is a typical and an economically important tree species for nut production with heterodichogamy. The absence of female and male flowering periods seriously affects both the pollination and fruit setting rates of walnuts, thereby affecting the yield and quality. Therefore, studying the characteristics and processes of flower bud differentiation helps in gaining a deeper understanding of the regularity of the mechanism of heterodichogamy in walnuts. In this study, a total of 3540 proteins were detected in walnut and 885 unique differentially expressed proteins (DEPs) were identified using the isobaric tags for the relative and absolute quantitation (iTRAQ)-labeling method. Among all DEPs, 12 common proteins were detected in all four of the obtained contrasts. GO and KEGG analyses of 12 common DEPs showed that their functions are distributed in the cytoplasm metabolic pathways, photosynthesis, glyoxylate and dicarboxylate metabolism, and the biosynthesis of secondary metabolites, which are involved in energy production and conversion, synthesis, and the breakdown of proteomes. In addition, a function analysis was performed, whereby the DEPs were classified as involved in photosynthesis, morphogenesis, metabolism, or the stress response. A total of eight proteins were identified as associated with the morphogenesis of stamen development, such as stamen-specific protein FIL1-like (XP_018830780.1), putative leucine-rich repeat receptor-like serine/threonine-protein kinase At2g24130 (XP_018822513.1), cytochrome P450 704B1-like isoform X2 (XP_018845266.1), ervatamin-B-like (XP_018824181.1), probable glucan endo-1,3-beta-glucosidase A6 (XP_018844051.1), pathogenesis-related protein 5-like (XP_018835774.1), GDSL esterase/lipase At5g22810-like (XP_018833146.1), and fatty acyl-CoA reductase 2 (XP_018848853.1). Our results predict several crucial proteins and deepen the understanding of the biochemical mechanism that regulates the formation of male and female flower buds in walnuts.

Morphogenesis, megagametogenesis, and microgametogenesis in Actinidia arguta flower buds

In dioecious plants, understanding sexual differentiation is important for improving cultivation techniques. Flower bud morphogenesis and gametophyte formation underlie the mechanism of sexual differentiation. Actinidia arguta (hardy kiwi) is a unisexual plant, and there have been few studies on its flower bud differentiation or gametophyte formation. We studied flower bud morphogenesis and gametophyte formation in A. arguta using paraffin sections and found that the morphogenesis of male and female flower buds was divided into six stages: the undifferentiated stage, inflorescence initials differentiation stage, calyx primordium differentiation stage, petal primordium differentiation stage, stamen primordium differentiation stage, and pistil primordium differentiation stage. Morphogenesis in the male inflorescence can reach quaternary flower primordia, and morphogenesis in the female inflorescence can reach the tertiary flower primordia, however, no Stage 4 lateral flowers in the male inflorescence or Stage 3 lateral flowers in the female inflorescence were found in natural situation. The embryo sac in female plants was a monocellular Polygonum type, and the embryo sac development in male plants was incomplete; abortion occurred before the archesporial cells developed. The male gametophyte differentiation process in the male plant was normal, with pollen grains similar in shape to a rugby ball and arranged neatly in the anther. The pollen grain differentiation in female plants was abnormal; abortion occurred at the uninucleate microspore stage, and mature pollen grains were irregularly spherical.

Positive selection and relaxed purifying selection contribute to rapid evolution of male-biased genes in a dioecious flowering plant.

Sex-biased genes offer insights into the evolution of sexual dimorphism. Sex-biased genes, especially those with male bias, show elevated evolutionary rates of protein sequences driven by positive selection and relaxed purifying selection in animals. Although rapid sequence evolution of sex-biased genes and evolutionary forces have been investigated in animals and brown algae, less is known about evolutionary forces in dioecious angiosperms. In this study, we separately compared the expression of sex-biased genes between female and male floral buds and between female and male flowers at anthesis in dioecious Trichosanthes pilosa (Cucurbitaceae). In floral buds, sex-biased gene expression was pervasive, and had significantly different roles in sexual dimorphism such as physiology. We observed higher rates of sequence evolution for male-biased genes in floral buds compared to female-biased and unbiased genes. Male-biased genes under positive selection were mainly associated with functions to abiotic stress and immune responses, suggesting that high evolutionary rates are driven by adaptive evolution. Additionally, relaxed purifying selection may contribute to accelerated evolution in male-biased genes generated by gene duplication. Our findings, for the first time in angiosperms, suggest evident rapid evolution of male-biased genes, advance our understanding of the patterns and forces driving the evolution of sexual dimorphism in dioecious plants.

Four new species of Zeugodacus Hendel (Diptera, Tephritidae, Dacinae, Dacini) and new records of dacines from India.

Four new species of Zeugodacus Hendel are described from India viz., Zeugodacusmomordicae David & Ajaykumara, sp. nov. from Arunachal Pradesh infesting male flower buds of Momordicadioica, Zeugodacusnasivittatus David & Abhishek, sp. nov. from Meghalaya, Zeugodacus (Sinodacus) sinuvittatus David & Abhishek, sp. nov. from Himachal Pradesh and Zeugodacus (Zeugodacus) umiam David & Kennedy, sp. nov. from Meghalaya. An illustrated key to all species of Zeugodacus from India is also included. Bactrocera (Parazeugodacus) abbreviata (Hardy) and Dacus (Mellesis) vijaysegarani Drew & Hancock are recorded for the first time from India.

High temperature treatment induced production of unreduced 2n pollen in Camellia oleifera

Unreduced gametes through chromosome doubling play a major role in the process of plant polyploidization. Our previous work confirmed that Camellia oleifera can produce natural 2n pollen, and it is possible to induce the 2n pollen formation by high temperature treatment. This study focused on the optimization of the 2n pollen induction technique and the mechanisms of high temperature-induced 2n pollen formation in C. oleifera. We found that the optimal protocol for inducing 2n pollen via high temperature was to perform 45 °C with 4 h at the prophase I stage of the pollen mother cells (PMCs). Meanwhile, high temperature significantly decreased the yield and fertility of 2n pollen. Through the observation of meiosis, abnormal chromosome and cytological behaviour was discovered under high-temperature treatment, and we confirmed that the formation of 2n pollen is caused by abnormal cell plate. Based on weighted gene co-expression network analysis, fifteen hub genes related to cell cycle control were identified. After male flower buds were exposed to heat shock, polygalacturonase gene (CoPGX3) was significantly upregulated. We inferred that high temperature causes the CoPGX3 gene to be overexpressed and that CoPGX3 is redistributed into the cytosol where it degrades cytoplasmic pectin, which leads to an abnormal cell plate. Furthermore, abnormal cytokinesis resulted in the formation of dyads and triads, and PMCs divided to produce 2n pollen. Our findings provide new insights into the mechanism of 2n pollen induced by high temperature in a woody plant and lay a foundation for further ploidy breeding of C. oleifera.

Uma nova abordagem para micropropagação de begônias por explantes de botões florais

Abstract Begonias grown in greenhouses are susceptible to devastating disease caused by pathogenic bacteria and fungi, decreasing the quality of propagated material. Plant tissue culture provides an alternative for rapid propagation of healthy Begonia material. The present study was undertaken to develop the protocol of micropropagation of three Begonia species and one hybrid from inflorescence explants. Male flower buds with part of pedicel restricted to 1 mm have been cultured in vitro on 6 variants of modified N6 media. Adventitious shoot organogenesis has been shown to occur from both pedicel and receptacle tissues under the action of any type of cytokinin applied, whereas BA and 2-iP triggered mainly the direct organogenesis, while TDZ proceeding morphogenic events through the stage of callus formation. For the culture establishment in vitro the most effective was the medium, supplemented with 1.5 µM 2-iP + 0.54 µM NAA with the addition of 40 mg L-1 adenine sulfate, contributed to the highest shoot regeneration from floral explants of all begonias studied. Histological analysis of adventitious buds pathways approved that their induction occurs under the treatment directly from the subepidermal cells. Morphological analysis performed after plantlets adaptation to the greenhouse conditions showed no morphological or bloom variations in the progeny, derived from the begonias inflorescence. The suggested technique considered as a practical step toward obtaining the uniform planting material for the propagation of economically valuable Begonia plants.

Reproductive biology of Populus deltoides Bartr.

The detailed knowledge on the reproductive biology of plants is required for their successful cultivation and conservation. The reproductive characteristics of trees are required to assess the diversity patterns and community structure of the forests. Understanding the reproductive characteristics, such as, pollination, breeding systems, phenology, and floral biology of Populus deltoides is immensely important; because it is one the most important tree species in India. The review documented the physiological basis of the reproductive characteristics of poplar tree. Literature indicated that the dioecious species shows a regular flowering patternin both the male and female trees, and they produces flower (catkin) every year. In all the species, male flower buds bursted earlier than the vegetative bud, whereas, female flower buds bursted after the emergence of leaves. Unisexual flowering occurs between February and April in both male & female, and the dispersal of seeds reported in late May or early June. Most of the pollen grains exhibited reddish colour. The fruits are capsule shaped, splits into 3or 4 parts, and release cotton like seeds, which are viable for only a short period. Poplar mostly regenerates from seed under natural conditions, but are also capable of asexual or vegetative reproduction under artificial conditions. The male and female plant produces a significant number of hybrids seeds; resulting in an excellent growth of the plants. The species displayed cross-pollination characteristics. The information documented in the current literature will be quite useful to understand the reproductive characteristics of the Populus deltoides.

Determination of the Microspore Development Concerning Floral Morphology for Improving Drought Tolerant Citron Watermelon (Citrullus lanatus var. citroides) Rootstocks Via Androgenesis.

The negative effects of abiotic stress factors (especially drought and salinity) that arise due to global climate change are increasing in today’s agricultural production. To eliminate the negative effects of these stress factors, some cultural practices have been implemented such as the breeding of tolerant cultivars and rootstock, exogenous applications of PGPR, AMF, and chemical substances, etc. Contrary to exogenous applications, providing tolerance with breeding methods has an important role in efficient and sustainable agriculture. Androgenesis is a phenomenon for the induction and development of microspore-derived pure lines, as initial material for F1 cultivar breeding, in different vegetable crops. Success in microspore embryogenesis is considerably related to the culture of microspores at an appropriate developmental stage. There is no standard protocol for the anther culture in citron watermelon (Citrullus lanatus var. citroides). Thus, a relation between floral morphology (bud length and width, bud index, sepal position, petal color), and microspore development (microspore diameter, uninucleated and binucleated phases) was investigated in citron. The male flower buds were collected at different sizes and microspore development was observed in both light and fluorescence microscopy. A strong positive relationship was detected between flower bud morphology and the specific stages of microspore development. To the author’s knowledge, this is the first report to indicate that the microspore developmental stage can be estimated by flower bud morphology, and applied in the anther/microspore culture of citron watermelon.

High Temperature-Induced Spindle Destabilization Results in Aborted Pollen Production in Populus

High temperature can induce the production of 2n gametes and aborted pollen during microsporogenesis in Populus canescens. However, the mechanism by which high temperature induces pollen abortion remains unknown. Here, pollen abortion was induced by exposing male flower buds of P. canescens to 38 and 41 °C; pollen morphology, meiotic abnormalities, defects of the meiotic microtubular cytoskeleton, and tapetum development were characterized. We found that the dominant meiotic stage, temperature, and duration of treatment significantly affected the percentage of high temperature-induced aborted pollen. Damaged spindle microtubules and depolymerized microtubular cytoskeletons were observed, which resulted in many lagging chromosomes at anaphase I and II as well as aneuploid male gametes and micronuclei, thereby generating aborted pollen grains. Tapetum disintegration was also delayed. The anther dehisced normally, and some viable pollen grains were released. However, no sufficient evidence showed that the delayed degradation of the tapetum was responsible for pollen abortion.

Production of double haploid watermelon via maternal haploid induction.

Production of double haploid watermelon via maternal haploid induction.

Transcriptional Regulation of Female and Male Flower Bud Initiation and Development in Pecan (Carya illinoensis)

Pecan (Carya illinoensis) nuts are delicious and rich in unsaturated fatty acids, which are beneficial for human health. Their yield is closely related to several factors, such as the ratio of female and male flowers. We sampled and paraffin-sectioned female and male flower buds for one year and determined the stages of initial flower bud differentiation, floral primordium formation, and pistil and stamen primordium formation. We then performed transcriptome sequencing on these stages. Our data analysis suggested that FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 play a role in flower bud differentiation. J3 was highly expressed in the early stage of female flower buds and may play a role in regulating flower bud differentiation and flowering time. Genes such as NF-YA1 and STM were expressed during male flower bud development. NF-YA1 belongs to the NF-Y transcription factor family and may initiate downstream events leading to floral transformation. STM promoted the transformation of leaf buds to flower buds. AP2 may have been involved in the establishment of floral meristem characteristics and the determination of floral organ characteristics. Our results lay a foundation for the control and subsequent regulation of female and male flower bud differentiation and yield improvement.

Identification and analysis of miRNAs differentially expressed in male and female Trichosanthes kirilowii maxim

Trichosanthes kirilowii Maxim. (TK) is a dioecious plant in the Cucurbitaceae family of which different sexes have separate medicinal uses. We used Illumina high-throughput sequencing technology to sequence miRNAs from male and female flower buds of TK. We performed bioinformatics analysis, miRNA identification, and target gene prediction on the data obtained from sequencing, and association analysis was performed in combination with the results of a previous transcriptome sequencing study. As a result, there were 80 differentially expressed miRNAs (DESs) between the female and male plants (48 upregulated and 32 downregulated in female plants). Moreover, 27 novel miRNAs in DESs were predicted to have 282 target genes, and 51 known miRNAs were predicted to have 3418 target genes. By establishing a regulatory network between miRNAs and target genes, 12 core genes were screened, including 7 miRNAs and 5 target genes. Among them, tkmiR157a-5p, tkmiR156c, tkmiR156_2, and tkmiR156k_2 jointly target the regulation of tkSPL18 and tkSPL13B. These two target genes are specifically expressed in male and female plants, respectively, and are involved in the biosynthesis process of BR, which is closely related to the sex differentiation process of TK. The identification of these miRNAs will provide a reference for the analysis of the sex differentiation mechanism of TK.

Expression patterns of ABCE model genes during flower development of melon (Cucumis melo L.)

In production, most cultivars of melon are andromonoecious and characterized by carrying both male and bisexual flowers on the same plant. In this study, four A-class genes (CmAP1a, CmAP1b, CmAP2a and CmAP2b), two B-class genes (CmAP3 and CmPI), two C-class genes (CmAGa and CmAGb) and four E-class genes (CmSEP1,2,3,4) were identified in melon. However, no D-class gene of melon was identified. The conserved domains of ABCE function proteins showed relatively high similarity between Arabidopsis and melon. The expression patterns of ABCE homeotic genes in different flower buds of melon suggested that transcripts of CmAP1a, CmPI and CmSEP1 in bisexual buds were significantly lower than that in male flower buds, while the expression levels of CmAGa, CmAGb and CmSEP4 in bisexual flower buds were significantly higher than that in male flower buds. There was no significant difference in expression levels of other ABCE model genes between male buds and bisexual buds. Subsequently, qRT-PCR was performed in different floral organs of bisexual flowers in melon. For A class genes, CmAP1a and CmAP1b showed the highest accumulation in sepals than petals, stamens and pistil, while CmAP2a and CmAP2b revealed the highest expression in pistil than other three floral organs. For B class genes, CmAP3 and CmPI were highly accumulated in petals and stamens though CmAP3 also showed abundant accumulation in pistil. For C class genes, the expression levels of CmAGa and CmAGb were higher in stamens and pistil than that in sepals and petals. For E class genes, CmSEP1 showed higher expression level in sepals and petals than stamens and pistil. CmSEP2, CmSEP3 and CmSEP4 showed the highest accumulation in pistil than other floral organs. These results provided a theoretical basis for studying the function of ABCE homeotic genes in floral organs development of melon.

Gibberellins as a novel mutagen for inducing 2n gametes in plants.

The plant hormone gibberellin (GA) regulates many physiological processes, such as cell differentiation, cell elongation, seed germination, and the response to abiotic stress. Here, we found that injecting male flower buds with exogenous gibberellic acid (GA3) caused defects in meiotic cytokinesis by interfering with radial microtubule array formation resulting in meiotic restitution and 2n pollen production in Populus. A protocol for inducing 2n pollen in Populus with GA3 was established by investigating the effects of the dominant meiotic stage, GA3 concentration, and injection time. The dominant meiotic stage (F = 41.882, P < 0.001) and GA3 injection time (F = 172.466, P < 0.001) had significant effects on the frequency of induced 2n pollen. However, the GA3 concentration (F = 1.391, P = 0.253) did not have a significant effect on the frequency of induced 2n pollen. The highest frequency of GA3-induced 2n pollen (21.37%) was observed when the dominant meiotic stage of the pollen mother cells was prophase II and seven injections of 10 μM GA3 were given. Eighteen triploids were generated from GA3-induced 2n pollen. Thus, GA3 can be exploited as a novel mutagen to induce flowering plants to generate diploid male gametes. Our findings provide some new insight into the function of GAs in plants.

Two New Species of Mutualistic Gall Midges (Diptera: Cecidomyiidae) Responsible for the Pollination of Phyllanthus flexuosus (Phyllanthaceae) and Using Its Male Flower Buds as Brood-Sites

Two new species of gall midges (Diptera: Cecidomyiidae: Cecidomyiinae) associated with male flower buds of Phyllanthus flexuosus (Phyllanthaceae) are described and illustrated. Clinodiplosis gagnei Elsayed, new species, induces male flower bud galls, whereas Macrolabis katoi Elsayed, new species, is an inquiline. Females of both species visit the host plant at flowering in search of suitable oviposition sites and, in doing so, become dusted with pollen of the opened male flowers and come in contact with the stigmas of female flowers. Larvae of both gall midge species overwinter in the ground, and each has only one generation per year. This is the first report of the genus Macrolabis in Japan.

Comprehensive Identification and Analyses of the GRF Gene Family in the Whole-Genome of Four Juglandaceae Species.

The GRF gene family plays an important role in plant growth and development as regulators involved in plant hormone signaling and metabolism. However, the Juglandaceae GRF gene family remains to be studied. Here, we identified 15, 15, 19, and 20 GRF genes in J. regia, C. illinoinensis, J. sigillata, and J. mandshurica, respectively. The phylogeny shows that the Juglandaceae family GRF is divided into two subfamilies, the ε-group and the non-ε-group, and that selection pressure analysis did not detect amino acid loci subject to positive selection pressure. In addition, we found that the duplications of the Juglandaceae family GRF genes were all segmental duplication events, and a total of 79 orthologous gene pairs and one paralogous homologous gene pair were identified in four Juglandaceae families. The Ka/KS ratios between these homologous gene pairs were further analyzed, and the Ka/KS values were all less than 1, indicating that purifying selection plays an important role in the evolution of the Juglandaceae family GRF genes. The codon bias of genes in the GRF family of Juglandaceae species is weak, and is affected by both natural selection pressure and base mutation, and translation selection plays a dominant role in the mutation pressure in codon usage. Finally, expression analysis showed that GRF genes play important roles in pecan embryo development and walnut male and female flower bud development, but with different expression patterns. In conclusion, this study will serve as a rich genetic resource for exploring the molecular mechanisms of flower bud differentiation and embryo development in Juglandaceae. In addition, this is the first study to report the GRF gene family in the Juglandaceae family; therefore, our study will provide guidance for future comparative and functional genomic studies of the GRF gene family in the Juglandaceae specie.

A CLAVATA3-like Gene Acts as a Gynoecium Suppression Function in White Campion.

How do separate sexes originate and evolve? Plants provide many opportunities to address this question as they have diverse mating systems and separate sexes (dioecy) that evolved many times independently. The classic “two-factor” model for evolution of separate sexes proposes that males and females can evolve from hermaphrodites via the spread of male and female sterility mutations that turn hermaphrodites into females and males, respectively. This widely accepted model was inspired by early genetic work in dioecious white campion (Silene latifolia) that revealed the presence of two sex-determining factors on the Y-chromosome, though the actual genes remained unknown. Here, we report identification and functional analysis of the putative sex-determining gene in S. latifolia, corresponding to the gynoecium suppression factor (GSF). We demonstrate that GSF likely corresponds to a Y-linked CLV3-like gene that is specifically expressed in early male flower buds and encodes the protein that suppresses gynoecium development in S. latifolia. Interestingly, GSFY has a dysfunctional X-linked homolog (GSFX) and their synonymous divergence (dS = 17.9%) is consistent with the age of sex chromosomes in this species. We propose that female development in S. latifolia is controlled via the WUSCHEL-CLAVATA feedback loop, with the X-linked WUSCHEL-like and Y-linked CLV3-like genes, respectively. Evolution of dioecy in the S. latifolia ancestor likely involved inclusion of ancestral GSFY into the nonrecombining region on the nascent Y-chromosome and GSFX loss of function, which resulted in disbalance of the WUSCHEL-CLAVATA feedback loop between the sexes and ensured gynoecium suppression in males.

Pollination of Phyllanthus (Phyllanthaceae) by gall midges that use male flower buds as larval brood sites

Leafflowers (Phyllanthus; Phyllanthaceae) are engaged in obligate mutualisms with leafflower moths (Epicephala) that actively pollinate leafflowers and consume the developing seeds as larvae. Among the >1,000 species of leafflowers worldwide, however, the mutualism is considered to be present in only roughly half of the species. Knowledge on pollination biology of the remaining species is critical for understanding the origin of the mutualism, but such information is thus far limited. We studied the pollination biology of two Phyllanthus species in Japan (P. flexuosus and P. liukiuensis) and one species in New Caledonia (P. baladensis); the three species belong to three distantly related subgenera but share a characteristic dark red floral display. A bagging experiment showed that P. flexuosus is self-incompatible and wind hardly plays a role in pollination. In P. flexuosus, gall midges of the genera Clinodiplosis and Macrolabis were the most consistent and abundant flower visitors. They fed on nectar at the spongy floral disc of male flowers and laid eggs in male flower buds, picking up pollen and contacting the styles of nearby female flowers in the process. Infested buds developed into sterile galls, within which midge larvae completed their development. These results indicate that P. flexuosus is pollinated by gall midges that use male flower buds as larval brood sites. Because Macrolabis larvae infested galls at a later stage than did Clinodiplosis, Macrolabis is likely an inquiline in the gall induced by Clinodiplosis. Association with gall midges was also observed in P. liukiuensis and P. baladensis flowers, suggesting that pollination by gall midges likely occurs in at least three subgenera of Phyllanthus, two of which contain species pollinated by specialized leafflower moths.