Molecular Network for Regulation of Ovule Number in Plants.

In seed-bearing plants, the ovule ("small egg") is the organ within the gynoecium that develops into a seed after fertilization. The gynoecium located in the inner compartment of the flower turns into a fruit. The number of ovules in the ovary determines the upper limit or the potential of seed number per fruit in plants, greatly affecting the final seed yield. Ovule number is an important adaptive characteristic for plant evolution and an agronomic trait for crop improvement. Therefore, understanding the mechanism and pathways of ovule number regulation becomes a significant research aspect in plant science. This review summarizes the ovule number regulators and their regulatory mechanisms and pathways. Specially, an integrated molecular network for ovule number regulation is constructed, in which phytohormones played a central role, followed by transcription factors, enzymes, other protein and micro-RNA. Of them, AUX, BR and CK are positive regulator of ovule number, whereas GA acts negatively on it. Interestingly, many ovule number regulators have conserved functions across several plant taxa, which should be the targets of genetic improvement via breeding or gene editing. Many ovule number regulators identified to date are involved in the diverse biological process, such as ovule primordia formation, ovule initiation, patterning, and morphogenesis. The relations between ovule number and related characteristics/traits especially of gynoecium/fruit size, ovule fertility, and final seed number, as well as upcoming research questions, are also discussed. In summary, this review provides a general overview of the present finding in ovule number regulation, which represents a more comprehensive and in-depth cognition on it.

Mesozoic cupules and the origin of the angiosperm second integument.

The second integument of the angiosperm ovule is unique among seed plants, with developmental genetics that are distinct from those of the inner integument1. Understanding how the second integument should be compared to structures in other seed plants is therefore crucial to resolving the long-standing question of the origin of angiosperms2-6. Attention has focused on several extinct plants with recurved cupules that are reminiscent of the anatropous organization of the basic bitegmic ovules of angiosperms1-6, but interpretations have been hampered by inadequate information on the relevant fossils. Here we describe abundant exceptionally well-preserved recurved cupules from a newly discovered silicified peat dating to the Early Cretaceous epoch (around 125.6 million years ago) in Inner Mongolia, China. The new material, combined with re-examination of potentially related fossils, indicates that the recurved cupules of several groups of Mesozoic plants are all fundamentally comparable, and that their structure is consistent with the recurved form and development of the second integument in the bitegmic anatropous ovules of angiosperms. Recognition of these angiosperm relatives (angiophytes) provides a partial answer to the question of angiosperm origins, will help to focus future work on seed plant phylogenetics and has important implications for ideas on the origin of the angiosperm carpel.

Gynoecium with carpel dimorphism in Tricomaria usillo, comparison with other genera of the Carolus clade (Malpighiaceae).

We analyzed the gynoecium morphology and anatomy of Tricomaria usillo in young and mature flowers from diverse populations in order to analyze the differentiation of structure and function of the parts of the carpel. We also aimed to find the potential pollinators and associate the morphology of the gynoecium with its role. We compare the characteristics of the gynoecium of T. usillo and discuss the carpel dimorphism with other genera within the Carolus clade in relation with their pollination syndromes. Carpels were processed according to classic techniques for scanning electron microscopy and bright field microscopy. We conducted field observation in different populations of T. usillo and captured the insects that were identified to specific level. The gynoecium of T. usillo shows inter-population and intra-individual variability. Some have three well-developed carpels, while most of them present two posterior carpels with differentiated styles and stigmas and the anterior one with a shorter style with or without stigma. The ovary has three locules with one ovule each. A compitum is formed and all ovules may be fecundated. However, fruits have generally one seed that develops in the anterior locule. Centris brethesi is the potential pollinator. The gynoecium of T. usillo reflects part of the variation in the carpel dimorphism that probably arose in the branch of the Carolus clade, and evolved in diverse ways in the lineages of this group. Tricomaria usillo seems to represent a recent transition towards reaching a stable form of carpel dimorphism and definitive division of labors of the carpels.

Development of the ovule and seed of Habanero chili pepper (Capsicum chinense Jacq.): Anatomical characterization and immunocytochemical patterns of pectin methyl-esterification.

Ovule and seed development in plants has long fascinated the scientific community given the complex cell coordination implicated in these processes. These cell events are highly conserved but are not necessarily representative of all plants. In this study, with the aim of obtaining information regarding the cellular patterns that follow the usual development of the ovule and the zygotic embryo, we carried out an integral anatomical study of the Capsicum chinense Jacq., floral buds and seeds at various days during maturation. This study allowed us to identify the main histo-morphological stages accompanying the transition of somatic cells into the macrospore, female gamete, and the zygotic embryogenesis. This knowledge is fundamental for future biotechnological research focused on solving the morphological recalcitrance observed during the in vitro induction of somatic or microspore embryogenesis in Capsicum. For the first time in C. chinense, we have described the hypostases, a putative source of plant growth regulators, and "the corrosion cavity", a space around the embryo. Additionally, the cell wall pectin-esterification status was investigated by immunohistology. At early stages of morphogenesis, the pectin is highly methyl-esterified; however, methyl-esterification decreases gradually throughout the process. A comparison of the results obtained here, together with the histo- and immunological changes occurring during the somatic and microspore embryogenesis, should help to elucidate the biochemical mechanisms that trigger the morphogenic events in Capsicum spp.

Effect of flower orientation on the male and female traits of Myrtillocactus geometrizans (Cactaceae).

Intra-individual variation in the production and size of reproductive traits has been documented in columnar cacti, being higher in equator-facing flowers. Such variation is attributed to the high amount of PAR intercepted by stems oriented towards the equator. Most studies focused on this phenomenon have documented the existence of intra-individual variation on traits associated with the female function; however, its impact on traits associated with the male function has been neglected. We tested the hypothesis that equator-facing flowers of Myrtillocactus geometrizans exhibit higher values on traits associated with both male and female functions than flowers facing against it. Number and size of anthers and ovaries, pollen:ovule ratio and number and quality of pollen grains (diameter, germinability, viability and pollen tube length) were estimated from reproductive structures facing north and south, and compared with t-tests between orientations. Number of anthers per flower, number of pollen grains per anther and per floral bud; pollen size, viability and germinability; pollen tube length; ovary length and pollen:ovule ratio were significantly higher in reproductive structures oriented towards the south (i.e. equator). These findings suggest that intra-individual variation in floral traits of M. geometrizans might be associated with different availability of resources in branches with contrasting orientation. Our results provide new evidence of the existence of a response to an orientation-dependent extrinsic gradient. To our knowledge, this is the first study documenting the existence of intra-individual variation on pollen quality and P:O ratio in Cactaceae species.

Egg activation-triggered shape change in the Dictyota dichotoma (Phaeophyceae) zygote is actin-myosin and secretion dependent.

Background and Aims: Cellular morphogenesis in land plants and brown algae is typically a slow process involving growth established by an interplay of turgor pressure and cell wall rigidity. However, a recent study showed that zygotes of the brown alga Dictyota dichotoma undergo a rapid shape change from a sphere to an elongated spheroid in about 90 s, establishing the first body axis. Methods: Using a combination of pharmacology, staining techniques, membrane depolarization and microscopy techniques (brightfield, transmission electron microscopy and confocal laser scanning microscopy), egg activation and the shape change of the egg cell of D. dichotoma was studied. Key Results: It was established that elongation of the zygote does not involve growth, i.e. a positive change in size. The elongation is dependent on F-actin and myosin but independent of microtubules. Secretion was also found to be necessary for elongation after addition of brefeldin A. Moreover, a temporal correlation between extracellular matrix secretion and elongation was observed. Ionomycin and high potassium seawater are capable of triggering the onset of elongation, suggesting a role for membrane depolarization and calcium influx in the signalling mechanism. The elongated cells are shorter in the presence of ionomycin, suggesting a role for calcium in elongation. Conclusions: A model is proposed in which the fast elongation of the fertilized egg in Dictyota is accomplished by a force generated by F-actin and myosin, regulated by cytoplasmic calcium concentrations and by secretion during elongation lowering the antagonistic force. The finding of early extracellular matrix secretion, membrane depolarization and ionophore-triggered egg activation suggest significant differences in the mechanism of egg activation signalling between D. dichotoma and the oogamous brown algal model system Fucus .

Developmental landmarks during floral ontogeny of jalapeño chili pepper (Capsicum annuum L.) and the effect of gibberellin on ovary growth.

Pepper (Capsicum annuum L.) is an important horticultural crop in many regions of the world. The final shape and size of the fruit are known to be determined at a very early step of flower development. During flower development hormonal treatments using gibberellins seem to promote growth resulting in higher yield and fruit quality. However, the morphological changes that occur in the pepper flowers after these treatments are largely unknown. In the present study, we provide a description of floral development landmarks of jalapeño chili pepper (cultivar Huichol), divided in nine representative stages from its initiation until the opening of the bud. We established a correlation among external flower development and the time and pattern of reproductive organogenesis. Male and female gametogenesis progression was used to define specific landmarks during flower maturation. The pattern of expression of key genes involved in gibberellin metabolism and response was also evaluated in the nine flower stages. The proposed development framework was used to analyze the effect of gibberellin treatments in the development of the flower. We observed both an effect of the treatment in the histology of the ovary tissue and an increase in the level of expression of CaGA2ox1 and CaGID1b genes. The developmental stages we defined for this species are very useful to analyze the molecular and morphological changes after hormonal treatments.

Further study of Late Devonian seed plant Cosmosperma polyloba: its reconstruction and evolutionary significance.

BACKGROUND: The earliest seed plants in the Late Devonian (Famennian) are abundant and well known. However, most of them lack information regarding the frond system and reconstruction. Cosmosperma polyloba represents the first Devonian ovule in China and East Asia, and its cupules, isolated synangiate pollen organs and pinnules have been studied in the preceding years. RESULTS: New fossils of Cosmosperma were obtained from the type locality, i.e. the Leigutai Member of the Wutong Formation in Fanwan Village, Changxing County, Zhejiang Province, South China. The collection illustrates stems and fronds extensively covered in prickles, as well as fertile portions including uniovulate cupules and anisotomous branches bearing synangiate pollen organs. The stems are unbranched and bear fronds helically. Fronds are dimorphic, displaying bifurcate and trifurcate types, with the latter possibly connected to fertile rachises terminated by pollen organs. Tertiary and quaternary rachises possessing pinnules are arranged alternately (pinnately). The cupule is uniovulate and the ovule has four linear integumentary lobes fused in basal 1/3. The striations on the stems and rachises may indicate a Sparganum-type cortex. CONCLUSIONS: Cosmosperma further demonstrates diversification of frond branching patterns in the earliest seed plants. The less-fused cupule and integument of this plant are considered primitive among Devonian spermatophytes with uniovulate cupules. We tentatively reconstructed Cosmosperma with an upright, semi-self-supporting habit, and the prickles along stems and frond rachises were interpreted as characteristics facilitating supporting rather than defensive structures.

Ovule positions within linear fruit are correlated with nonrandom mating in Robinia pseudoacacia.

Post-pollination processes can lead to nonrandom mating among compatible pollen donors. Moreover, morphological patterns of ovule development within linear fruits are reportedly nonrandom and depend on ovule position. However, little is known about the relationship between nonrandom mating and ovule position within linear fruit. Here, we combined controlled pollen competition experiments and paternity analyses on R. pseudoacacia to better understand nonrandom mating and its connection with ovule position. Molecular determination of siring success showed a significant departure from the expected ratio based on each kind of pollen mixture, suggesting a nonrandom mating. Outcrossed pollen grains, which were strongly favored, produced significantly more progeny than other pollen grains. Paternity analyses further revealed that the distribution of offspring produced by one specific pollen source was also nonrandom within linear fruit. The stylar end, which has a higher probability of maturation, produced a significantly higher number of outcrossed offspring than other offspring, suggesting a correlation between pollen source and ovule position. Our results suggested that a superior ovule position exists within the linear fruit in R. pseudoacacia, and the pollen that was strongly favored often preferentially occupies the ovules that were situated in a superior position, which ensured siring success and facilitated nonrandom mating.

Early Cretaceous Umkomasia from Mongolia: implications for homology of corystosperm cupules.

Corystosperms, a key extinct group of Late Permian to Early Cretaceous plants, are important for understanding seed plant phylogeny, including the evolution of the angiosperm carpel and anatropous bitegmic ovule. Here, we describe a new species of corystosperm seed-bearing organ, Umkomasia mongolica sp. nov., based on hundreds of three-dimensionally preserved mesofossils from the Early Cretaceous of Mongolia. Individual seed-bearing units of U. mongolica consist of a bract subtending an axis that bifurcates, with each fork (cupule stalk) bearing a cupule near the tip. Each cupule is formed by the strongly reflexed cupule stalk and two lateral flaps that partially enclose an erect seed. The seed is borne at, or close to, the tip of the reflexed cupule stalk, with the micropyle oriented towards the stalk base. The corystosperm cupule is generally interpreted as a modified leaf that bears a seed on its abaxial surface. However, U. mongolica suggests that an earlier interpretation, in which the seed is borne directly on an axis (shoot), is equally likely. The 'axial' interpretation suggests a possible relationship of corystosperms to Ginkgo. It also suggests that the cupules of corystosperms may be less distinct from those of Caytonia than has previously been supposed.

Transition from two to one integument in Prunus species: expression pattern of INNER NO OUTER (INO), ABERRANT TESTA SHAPE (ATS) and ETTIN (ETT).

While gymnosperm ovules have one integument, in most angiosperms two integuments surround the ovules. Unitegmic ovules have arisen independently several times during the evolution of angiosperms, but the ultimate genetic cause of the presence of a single integument remains elusive. We compared species of the genus Prunus that have different numbers of integuments: bitegmic species, such as Prunus armeniaca (apricot) and Prunus persica (peach), and unitegmic species, such as Prunus incisa, analyzing the expression pattern of genes that are involved in integument development in Arabidopsis thaliana: INNER NO OUTER (INO), ABERRANT TESTA SHAPE (ATS) and ETTIN (ETT). Bitegmic and unitegmic species showed similar INO expression patterns, indicative of the conservation of an outer integument. However, expression of ETT, which occurs in the boundary of the outer and inner integuments, was altered in unitegmic ovules, which showed lack of ETT expression. These results strongly suggest that the presence of a single integument could be attributable to the amalgamation of two integuments and support the role of ETT in the fusion of the outer and inner integuments in unitegmic ovules, a situation that could be widespread in other unitegmic species of angiosperms.

Integration and macroevolutionary patterns in the pollination biology of conifers.

Integration influences patterns of trait evolution, but the relationship between these patterns and the degree of trait integration is not well understood. To explore this further, we study a specialized pollination mechanism in conifers whose traits are linked through function but not development. This mechanism depends on interactions among three characters: pollen that is buoyant, ovules that face downward at pollination, and the production of a liquid droplet that buoyant grains float through to enter the ovule. We use a well-sampled phylogeny of conifers to test correlated evolution among these characters and specific sequences of character change. Using likelihood models of character evolution, we find that pollen morphology and ovule characters evolve in a concerted manner, where the flotation mechanism breaks down irreversibly following changes in orientation or drop production. The breakdown of this functional constraint, which may be facilitated by the lack of developmental integration among the constituent traits, is associated with increased trait variation and more diverse pollination strategies. Although this functional "release" increases diversity in some ways, the irreversible way in which the flotation mechanism is lost may eventually result in its complete disappearance from seed plant reproductive biology.

Structural and developmental variability in the female gametophyte of Griffithella hookeriana, Polypleurum stylosum, and Zeylanidium lichenoides and its bearing on the occurrence of single fertilization in Podostemaceae.

Angiosperms are characterized by the phenomenon of double fertilization with Podostemaceae as an exception that appears to extend to the entire family. Our earlier work demonstrated the cause of failure of double fertilization and ascertained the occurrence of single fertilization in Dalzellia zeylanica (Tristichoideae, Podostemaceae). In continuation with this work, three more members, i.e., Griffithella hookeriana (Tul.) Warming, Polypleurum stylosum (Wight) Hall, and Zeylanidium lichenoides (Kurz) Engl. (Podostemoideae), have been investigated in the present work. We studied the ontogenetic development of female gametophyte and tracked the path of the two sperm cells from the time of their formation in the pollen tube through their entry into the synergid and gamete fusion. We report the occurrence of a remarkably reduced 3-nucleate, 3-celled mature female gametophyte consisting of an egg cell and two synergids in all the three genera. Interestingly, the central cell is formed during female gametophyte development, but exhibits a species-specific, limited life span, and eventually degenerates prior to the entry of the pollen tube into the synergid, resulting in a failure of double fertilization. Sperm dimorphism on the basis of fluorochrome stainability has been recorded in Z. lichenoides. Further, morphogenetic constraints on the part of male (sperm selection, functional reductionism) and female gametophyte (structural reductionism, inaccessibility of central cell) presumably ensure the failure of double fertilization in these species. Thus, loss of double fertilization in this family is likely a derived condition.

Frequency-dependent pollinator discrimination acts against female plants in the gynodioecious Geranium maculatum.

BACKGROUND AND AIMS: Gynodioecy, the co-occurrence of female and hermaphroditic individuals, is thought to be an intermediate step between hermaphroditism and separate sexes, a major transition in flowering plants. Because retaining females in a population requires that they have increased seed fitness (to compensate for the lack of pollen fitness), factors that affect seed fitness are of great importance to the evolution of this mating system and have often been studied. However, factors negatively affecting female fitness are equally important and have been largely neglected. One such factor stems from female flowers being less attractive to insects than hermaphrodite flowers, thereby decreasing their relative fitness. METHODS: To test the severity and consequences of this type of pollinator discrimination in Geranium maculatum, experimental populations with the range of sex ratios observed in nature were created, ranging from 13 % to 42 % females. Pollinators were observed in order to measure the strength of discrimination, and pollen deposition and seed production of both sexes were measured to determine the fitness consequences of this discrimination. Additionally a comparison was made across the sex ratios to determine whether discrimination was frequency-dependent. KEY RESULTS: It was found that female flowers, on average, were visited at half of the rate of hermaphrodite flowers, which decreased their pollen receipt and seed production. Additionally, females were most discriminated against when rare, due to both changes in the pollinators' behaviour and a shift in pollinator composition. CONCLUSIONS: The results suggest that pollinator discrimination negatively affects females' relative fitness when they are rare. Thus, the initial spread of females in a population, the first step in the evolution of gynodioecy, may be made more difficult due to pollinator discrimination.

Floral structure of Emmotum (Icacinaceae sensu stricto or Emmotaceae), a phylogenetically isolated genus of lamiids with a unique pseudotrimerous gynoecium, bitegmic ovules and monosporangiate thecae.

BACKGROUND AND AIMS: Icacinaceae sensu stricto consist of a group of early branching lineages of lamiids whose relationships are not yet resolved and whose detailed floral morphology is poorly known. The most bizarre flowers occur in Emmotum: the gynoecium has three locules on one side and none on the other. It has been interpreted as consisting of three fertile and two sterile carpels or of one fertile carpel with two longitudinal septa and two sterile carpels. This study focused primarily on the outer and inner morphology of the gynoecium to resolve its disputed structure, and ovule structure was also studied. In addition, the perianth and androecium were investigated. METHODS: Flowers and floral buds of two Emmotum species, E. harleyi and E. nitens, were collected and fixed in the field, and then studied by scanning electron microscopy. Microtome section series were used to reconstruct their morphology. KEY RESULTS: The gynoecium in Emmotum was confirmed as pentamerous, consisting of three fertile and two sterile carpels. Each of the three locules behaves as the single locule in other Icacinaceae, with the placenta of the two ovules being identical, which shows that three fertile carpels are present. In addition, it was found that the ovules are bitegmic, which is almost unique in lamiids, and that the stamens have monosporangiate thecae, which also occurs in the closely related family Oncothecaceae, but is not known from any other Icacinaceae sensu lato so far. CONCLUSIONS: The flowers of Emmotum have unique characters at different evolutionary levels: the pseudotrimerous gynoecium at angiosperm level, the bitegmic ovules at lamiid level and the monosporangiate thecae at family or family group level. However, in general, the floral morphology of Emmotum fits well in Icacinaceae. More comparative research on flower structure is necessary in Icacinaceae and other early branching lineages of lamiids to better understand the initial evolution of this large lineage of asterids.

Floral structure and development in the monoecious palm Gaussia attenuata (Arecaceae; Arecoideae).

BACKGROUND AND AIMS: Sexual dimorphism, at both the flower and plant level, is widespread in the palm family (Arecaceae), in contrast to the situation in angiosperms as a whole. The tribe Chamaedoreeae is of special interest for studies of the evolution of sexual expression since dioecy appears to have evolved independently twice in this group from a monoecious ancestor. In order to understand the underlying evolutionary pathways, it is important to obtain detailed information on flower structure and development in each of the main clades. METHODS: Dissection and light and scanning electron microscopy were performed on developing flowers of Gaussia attenuata, a neotropical species belonging to one of the three monoecious genera of the tribe. KEY RESULTS: Like species of the other monoecious genera of the Chamaedoreeae (namely Hyophorbe and Synechanthus), G. attenuata produces a bisexual flower cluster known as an acervulus, consisting of a row of male flowers with a basal female flower. Whereas the sterile androecium of female flowers terminated its development at an early stage of floral ontogeny, the pistillode of male flowers was large in size but with no recognizable ovule, developing for a longer period of time. Conspicuous nectary differentiation in the pistillode suggested a possible role in pollinator attraction. CONCLUSIONS: Gaussia attenuata displays a number of floral characters that are likely to be ancestral to the tribe, notably the acervulus flower cluster, which is conserved in the other monoecious genera and also (albeit in a unisexual male form) in the dioecious genera (Wendlandiella and a few species of Chamaedorea). Comparison with earlier data from other genera suggests that large nectariferous pistillodes and early arrest in staminode development might also be regarded as ancestral characters in this tribe.

Temporal variation in phenotypic gender and expected functional gender within and among individuals in an annual plant.

BACKGROUND AND AIMS: Adaptive explanations for variation in sex allocation centre on variation in resource status and variation in the mating environment. The latter can occur when dichogamy causes siring opportunity to vary across the flowering season. In this study, it is hypothesized that the widespread tendency towards declining fruit-set from first to last flowers on plants can similarly lead to a varying mating environment by causing a temporal shift in the quality (not quantity) of siring opportunities. METHODS: A numerical model was developed to examine the effects of declining fruit-set on the expected male versus female reproductive success (functional gender) of first and last flowers on plants, and of early- and late-flowering plants. Within- and among-plant temporal variation in pollen production, ovule production and fruit-set in 70 Brassica rapa plants was then characterized to determine if trends in male and female investment mirror expected trends in functional gender. KEY RESULTS: Under a wide range of model conditions, functional femaleness decreased sharply in the last flowers on plants, and increased from early- to late-flowering plants in the population. In B. rapa, pollen production decreased more rapidly than ovule production from first to last flowers, leading to a within-plant increase in phenotypic femaleness. Among plants, ovule production decreased from early- to late-flowering plants, causing a temporal decrease in phenotypic femaleness. CONCLUSIONS: The numerical model confirmed that declining fruit-set can drive temporal variation in functional gender, especially among plants. The discrepancy between observed trends in phenotypic gender in B. rapa and expected functional gender predicted by the numerical model does not rule out the possibility that male reproductive success decreases with later flowering onset. If so, plants may experience selection for early flowering through male fitness.

Anatomical studies of ovule development in the post-bloom pistils of the 'Zuili' plum (Prunus salicina Lindl.).

In order to investigate the cause of poor fruit set in 'Zuili' plums, anatomical examinations of post-bloom pistils were conducted and the dates of young fruit drop were recorded during the growing seasons of 2008 and 2009. Pistils of cv. 'Black Amber' were also examined as an abundant setting control. Two major dropping periods were detected in 'Zuili': one during the first 5 d after full bloom (DAF) and another between 10 and 17 DAF. Anatomical analyses of the pistils at the full bloom stage revealed that half of the ovules had not developed embryos, which may have caused their early drop. In most dropped pistils collected at 17 DAF, the micropyle had not been penetrated by a pollen tube, indicating that they were not fertilized. 'Zuili' ovules initiated embryo division at 10-12 DAF, although thereafter embryo development was retarded when compared to the rates observed in 'Black Amber'. Ovule fertilization failure and inactive embryo development after ovule fertilization may be the major causes of the later fruit drop observed in 'Zuili' plum trees.

Advances in the floral structural characterization of the major subclades of Malpighiales, one of the largest orders of flowering plants.

BACKGROUND AND AIMS: Malpighiales are one of the largest angiosperm orders and have undergone radical systematic restructuring based on molecular phylogenetic studies. The clade has been recalcitrant to molecular phylogenetic reconstruction, but has become much more resolved at the suprafamilial level. It now contains so many newly identified clades that there is an urgent need for comparative studies to understand their structure, biology and evolution. This is especially true because the order contains a disproportionally large diversity of rain forest species and includes numerous agriculturally important plants. This study is a first broad systematic step in this endeavour. It focuses on a comparative structural overview of the flowers across all recently identified suprafamilial clades of Malpighiales, and points towards areas that desperately need attention. METHODS: The phylogenetic comparative analysis of floral structure for the order is based on our previously published studies on four suprafamilial clades of Malpighiales, including also four related rosid orders (Celastrales, Crossosomatales, Cucurbitales, Oxalidales). In addition, the results are compiled from a survey of over 3000 publications on macrosystematics, floral structure and embryology across all orders of the core eudicots. KEY RESULTS: Most new suprafamilial clades within Malpighiales are well supported by floral structural features. Inner morphological structures of the gynoecium (i.e. stigmatic lobes, inner shape of the locules, placentation, presence of obturators) and ovules (i.e. structure of the nucellus, thickness of the integuments, presence of vascular bundles in the integuments, presence of an endothelium in the inner integument) appear to be especially suitable for characterizing suprafamilial clades within Malpighiales. CONCLUSIONS: Although the current phylogenetic reconstruction of Malpighiales is much improved compared with earlier versions, it is incomplete, and further focused phylogenetic and morphological studies are needed. Once all major subclades of Malpighiales are elucidated, more in-depth studies on promising structural features can be conducted. In addition, once the phylogenetic tree of Malpighiales, including closely related orders, is more fully resolved, character optimization studies will be possible to reconstruct evolution of structural and biological features within the order.

Evolutionarily stable size of a megagametophyte: evolution of tiny megagametophytes of angiosperms from large ones of gymnosperms.

To examine the factors favoring large megagametophytes of gymnosperms and tiny ones of angiosperms, a game model for seed production was developed in which megagametophytes growing in the same female parent compete for resources provided by the parent. In the model, megagametophytes may continue to grow until seed completion or may cease to grow at a certain time and regrow at pollination or fertilization. Autonomous abortion of unpollinated or unfertilized megagametophytes may occur either at pollination or fertilization. Those megagametophytes absorb a certain amount of resources before abortion, due to constraints in the signal process, in addition to the resources absorbed before pollination or fertilization. It was found that both growth habits can be the ESS: megagametophytes continue to grow without cessation and monopolize resources, such as gymnosperms, or cease to grow until fertilization to reduce the loss of resources due to autonomous abortion, such as angiosperms. The former and the latter are the ESS if the time interval between pollination and fertilization is long and short, respectively. Thus, the fertilization interval may be a critical factor selecting for large megagametophytes of gymnosperms or tiny ones of angiosperms.