To attract or to repel? Diversity, evolution and role of the “most peculiar organ” in the Cuscuta flower (dodder, Convolvulaceae)—the infrastaminal scales

Cuscuta (dodders) is the only genus in Convolvulaceae and the Solanales more broadly in which corolla-stamen structures called infrastaminal scales (IFS) have reached a high degree of elaboration and diversification in a great number of species. Historically, morphological diversity of IFS has provided some of the most useful taxonomic characters at the species-level. However, their function has not been determined. We have performed a comparative study of the IFS in 147 Cuscuta taxa using light, scanning and transmission electron microscopy, and results were analyzed in relation to a Cuscuta phylogeny obtained from a combined analysis of nuclear ITS and plastid trnL-F sequences. The morphology and histochemistry of scales and/or trichomes on the staminal filaments were also examined in several other Convolvulaceae genera to provide a preliminary foundation for homology interpretation. To test the hypothesis that the role of IFS in Cuscuta is connected to sexual reproduction, we analyzed the correlations between scale characters and previously published values for pollen/ovule ratios and the number of stomata found in the nectary at the base of the ovary. While the correlations at the level of the entire genus were very low, subgeneric partitions revealed a strong relationship between scale characters and sexual reproduction indicators in subgenus Monogynella. However, this connection declined drastically in the subgenera Cuscuta and Grammica. Our results strongly suggest that scales in Cuscuta evolved in connection to a modification of their function in the flower: from nectar protection and holding in the first diverged subgenus Monogynella, to ovary/ovule protection against herbivorous insects in the derived subgenera Cuscuta and Grammica. Thus at least in the case of the latter subgenera, the protective/repellent role of the IFS may conflict with the attractant/rewarding function of the nectary found at the base of the ovary. In subgenus Monogynella, IFS fimbriae are similar to uniseriate glandular hairs, with the secretory cells entirely exposed. In the subgenera Grammica and Cuscuta, the fimbriae become more complex, with an internal distal laticifer, and a precise mechanism of latex release. Our study elaborates further on the development and ultrastructure of scales in C. gronovii, and provides details on the anatomy of the IFS in other species. The new morphology and micromorphology data confirm the significance of these structures for species-level systematics.     

Pollen evolution and its taxonomic significance in Cuscuta (dodders, Convolvulaceae)

The pollen morphology of 148 taxa (135 species and 13 varieties) of the parasitic plant genus Cuscuta (dodders, Convolvulaceae) was examined using scanning electron microscopy. Six quantitative characters were coded using the gap-weighting method and optimized onto a consensus tree constructed from three large-scale molecular phylogenies of the genus based on nuclear internal transcribed spacer (ITS) and plastid trn-LF sequences. The results indicate that 3-zonocolpate pollen is ancestral, while grains with more colpi (up to eight) have evolved only in two major lineages of Cuscuta (subg. Monogynella and clade O of subg. Grammica). Complex morphological intergradations occur between species when their tectum is described using the traditional qualitative types—imperforate, perforate, and microreticulate. This continuous variation is better expressed quantitatively as “percent perforation,” namely the proportion of perforated area (puncta or lumina) from the total tectum surface. Tectum imperforatum is likely the ancestral condition, while pollen grains with increasingly larger perforation areas have evolved multiple times. The reticulated tectum, unknown in other Convolvulaceae, has evolved in Cuscuta only in two lineages (subg. Monogynella, and clade O of subg. Grammica). Overall, the morphology of pollen supports Cuscuta as a sister to either the “bifid-style” Convolvulaceae clade (Dicranostyloideae) or to one of the members of this clade. Pollen characters alone are insufficient to reconstruct phylogenetic relationships; however, palynological information is useful for the species-level taxonomy of Cuscuta.     

Diversity and evolution of pollen-ovule production in <Emphasis Type="Italic">Cuscuta</Emphasis> (dodders,Convolvulaceae) in relation to floral morphology

Cuscuta (dodder, Convolvulaceae) is a genus of about 200 species of obligate stem parasites with subcosmopolitan distribution. The diversity of pollen and ovule production was surveyed in 128 species and ten varieties. Taxa were assigned to Cruden’s mating system categories based on their pollen-ovule ratios. Variation and correlations among floral characters were analyzed using regression and ANOVA, while the mating system categories were subjected to a linear discriminant and canonical variates analysis to assess their cohesiveness. Our data strongly suggest that most Cuscuta species possess a wide range of mixed-mating systems. Whereas four ovules develop in each flower, pollen production varies over three orders of magnitude. Several Cuscuta taxa are highly outcrossing, but no species could be identified that are exclusively selfing. The transition from the one-style flowers of subg. Monogynella to the two-style flowers of subgenera Cuscuta and Grammica, and from simultaneous to sequential maturation of the two stigmas in the latter subgenus, has decreased the role of herkogamy as a facilitator of outcrossing. These evolutionary changes are associated with an increase of species richness in subgenus Cuscuta, and especially in subgenus Grammica. Morphological features were not individually found to have a strong correlation to the mating system, but in general, larger corollas and stigmas were associated with greater pollen-ovule ratios. Cuscuta presents some puzzling results when considered in light of the sex allocation theory, as only some infrageneric lineages demonstrate the predicted pollen size-number tradeoff, while Cuscuta gracillima complex (in subgenus Grammica) displays an unexpected negative relationship between pollen size and style length. The relationship between host range and mating system is discussed, prompting further research into the co-evolution of pollination systems and life history traits between parasites and their host species.     

Diversity,evolution, and function of stomata bearing structures in Cuscuta (dodders,Convolvulaceae): From extrafloral nectar secretion to transpiration in arid conditions

Cuscuta includes ca. 200 species of functionally holoparasitic plants grouped in four subgenera: Monogynella, Cuscuta, Pachystigma, and Grammica. Multicellular structures with stomata in Cuscuta are represented by extrafloral nectaries (ENs), reported from the stems of one Monogynella species, and stomatiferous protuberances (SPs), which are non-secretory. These latter structures had been noted on the stems of three Grammica species more than a century ago but entirely forgotten until recently when similar, non-secretory SPs were reported on the flowers of several new Grammica species. Here we study for the first time: (1) the extent of occurrence, diversity and evolution of secretory (ENs) and non-secretory (SPs) multicellular structures in Cuscuta, and (2) the function of SPs. We undertook a character evolution study of ENs and SPs on the stems and flowers of 136 Cuscuta taxa, and examined the structure/ultrastructure of SPs. ENs are inferred as primitive and characterize subg. Monogynella. SPs are derived in the remaining subgenera; they are ubiquitous on the flowers of Cuscuta and Pachystigma, but absent on their stems. Subgenus Grammica species develop two functional types of stems during their life cycle: vegetative, exploratory stems with very low stomatal densities (and no SPs), and reproductive, haustorial stems with numerous SPs. Moreover, 24 species from nine clades of subg. Grammica have evolved morphologically diverse floral SPs with systematic significance. To preliminarily ascertain SP function, we determined in the field the water uptake of Tithonia tubiformis plants parasitized or not by Cuscuta costaricensis, a species with both stem and floral SPs, and the stomatal conductance of dodder stems and flowers, as well as host leaves. Water uptake of parasitized hosts was significantly higher compared to non-parasitized plants, even after host leaves were removed, both during the day and night. The increased water uptake of parasitized hosts and stomatal conductance values suggest a transpiration role for the SPs, which is also confirmed by their lacunar structure. Grammica species with floral SPs grow in arid areas or characterized by a pronounced dry season during flowering/fruiting, which suggests that SPs may have evolved to stimulate the host water uptake during these phenophases.     

Some further notes on the taxonomy and nomenclature ofCuscutaceae

The division ofCuscuta s.l. into 4 genera, based mainly on floral morphology, is corroborated by their embryology. Several new combinations inMonogynella andGrammica are proposed.     

Down the Slippery Slope: Plastid Genome Evolution in Convolvulaceae

Cuscuta (dodder) is the only parasitic genus found in Convolvulaceae (morning-glory family). We used long PCR approach to obtain large portions of plastid genome sequence from Cuscuta sandwichiana in order to determine the size, structure, gene content, and synteny in the plastid genome of this Cuscuta species belonging to the poorly investigated holoparasitic subgenus Grammica. These new sequences are compared with the tobacco chloroplast genome, and, where data are available, with corresponding regions from taxa in the other Cuscuta subgenera. When all known plastid genome structural rearrangements in parasitic and nonparasitic Convolvulaceae are considered in a molecular phylogenetic framework, three categories of rearrangements in Cuscuta are revealed: plesiomorphic, autapomorphic, and synapomorphic. Many of the changes in Cuscuta, previously attributed to its parasitic mode of life, are better explained either as plesiomorphic conditions within the family, i.e., conditions shared with the rest of the Convolvulaceae, or, in most cases, autapomorphies of particular Cuscuta taxa, not shared with the rest of the species in the genus. The synapomorphic rearrangements are most likely to correlate with the parasitic lifestyle, because they represent changes found in Cuscuta exclusively. However, it appears that most of the affected regions, belonging to all of these three categories, have probably no function (e.g., introns) or are of unknown function (a number of open reading frames, the function of which, if any, has yet to be discovered).[Reviewing Editor: Dr. Debashish Bhattacharya]     

Pollen tube reallocation in two preanthesis cleistogamous species, Ranalisma rostratum and Sagittaria guyanensis ssp. lappula (Alismataceae)

Pollen deposition on stigmas and pollen tube growth in two apocarpous species, Ranalisma rostratum and Sagittaria guyanensis ssp. lappula (Alismataceae), were examined with fluorescence microscopy. The reallocation of pollen tubes among pistils was observed in both species. The percentage of pollinated stigmas per flower was only 22.0% in R. rostratum and 51.0% in S. guyanensis, though the seed/ovule ratios are higher than 65% in both species. The number of pollen grains on each single stigma ranged from 0 to 96 in R. rostratum, and from 0 to 125 in S. guyanensis. When more than one pollen grain deposited on a stigma, all pollen tubes grew to the ovary, but only one of them turned towards the ovule and finally entered the nucleus. The other tubes grew through the receptacle tissue into ovules of adjacent carpels whose stigmas were unpollinated or pollinated later. The intercarpellary growth of pollen tubes could be a mechanism to increase the efficiency of sexual reproduction in an apocarpous gynoecium with low pollination on the pistils.     

Notes on the taxonomy ofCuscutaceae

The subdivision ofCuscutaceae into 4 genera (Cuscuta L. s. str.,Grammica Lour.,Monogynella Des Moul.,Kadurias Rafin.) is proposed.     

Pollen tubes enter neighbouring ovules by way of receptacle tissue,resulting in increased fruit-set in Sagittaria potamogetifolia Merr

Using fluorescence microscopy, deposition of pollen on stigmas and pollen tube growth in the gynoecium of Sagittaria potamogetifolia Merr., a monoecious species with an apocarpous gynoecium, were observed. The maximum rate of pollination averaged 83.9 +/- 4.7 %, and the number of pollen grains per stigma ranged from zero to 30. Pollen tubes grew through one stigma to the base of the ovary at almost the same speed, but generally only one of the pollen tubes then turned towards the ovule and finally entered the nucellus through the micropyle. The other pollen tubes grew through the ovary base and the receptacle tissue into ovules of adjacent carpels whose stigmas were not pollinated or which had been pollinated later. This phenomenon is termed pollen tube 'reallocation' by the authors. To verify the direct effect of the phenomenon on fruit set, artificial pollination experiments were conducted in which two or more pollen grains were placed onto only one stigma in each gynoecium; frequently more than one fruitlet was obtained from each flower treated. The reallocation of pollen tubes among pistils in the gynoecium could effect fertilization of ovules of unpollinated pistils and lead to an increase in sexual reproduction efficiency. It would, to some extent, also increase pollen tube competition among pistils of the whole gynoecium.     

Morphological variation in pollen grains of <Emphasis Type="Italic">Mucuna</Emphasis> (Leguminosae): new biogeographic and evolutionary patterns

Mucuna comprises 105 species with an overall pantropical distribution and is divided into three subgenera: M. subg. Mucuna, M. subg. Stizolobium and M. subg. Macrocarpa. Although phylogenetic studies have supported the occurrence of three main clades, evolutionary relationships among them are not fully resolved. The objective of this study was to examine pollen grain morphology from representatives of all three subgenera and map these onto the phylogenetic trees generated by analysis of other characters. Pollen grain surface, form, size, and aperture number were compared. A Bayesian inference tree using matK sequences was constructed. The results indicate that the representatives of M. subg. Macrocarpa have the smallest pollen grains in the genus (a synapomorphic character here identified for this subgenus) and that species of subgenus Mucuna (those with umbelliform inflorescences) have the largest pollen grains. Additional morphological diversity of the pollen grain surface was noted: reticulate and/or micro-reticulate (in all three subgenera), perforate, gemmate or verrucose (only in M. subg. Mucuna). For all studied taxa, the pollen grains are triaperturate, except for two species of M. subg. Mucuna, which have tetraperturate pollen. The phylogenetic tree obtained using the matK marker resolved M. subg. Stizolobium as the earliest diverging lineage in Mucuna. Based on this phylogeny, a reticulate ornamentation pattern of the pollen surface may represent the ancestral state for the genus, while the larger pollen size and the foraminate, gemmate, and verrucose ornamentations are derived characteristics within the genus. These putative derived ornamentations have been observed only in neotropical species.     

Permanent stigma closure in Bignoniaceae: Mechanism and implications for fruit set in self-incompatible species

The Bignoniaceae possess sensitive bilobate stigmas that close after stimulation. We determined factors involved in stigma closure and reopening in four species of Bignoniaceae from the National Parks of Catimbau, Northeastern Brazil (Anemopaegma laeve, Arrabidaea limae, Jacaranda rugosa and Tabebuia impetiginosa). The study showed that any mechanical touch promoted the stigmatic closure. Only the deposition of viable con-specific pollen (self- and cross-pollen), however, maintained stigmas permanently closed. In Arrabidaea limae part of the stigmas reopened after self- and cross-pollination. After mechanical stimulation, deposition of pollen from other plant species, of dead con-specific pollen or of sand, the stigma lobes opened again after 38 to 68 min. The definitive closure may have a negative effect on the reproductive success of the involved plants, because the stigmas are permanently unavailable for pollen deposition. In self-incompatible species with mass-flowering blooming pattern, where pollinators promote high levels of geitonogamy, definitive stigma closure causes low fruit set.     

Dichogamy in Salicornieae species: establishment of floral sex phases and evaluation of their frequency and efficacy in four species

Dichogamy in Salicornieae species is not clearly described in literature, and its efficiency remains untested. In this work we aim to establish the pathway for flower development, the degree of interaction between the two sexes, and the efficiency in overcoming selfing. Flower development was studied in six populations of four Salicornieae species at the Odiel Marsh Natural Park (Huelva, SW Spain) by calculating the percentage of different floral phases established in accordance with the position of their stamens and stigmas. To study the sexual functionality of each phase, the lengths of the perianth, stigma, and ovule were measured, and the number of germinated pollen grains on the stigmas was counted. In the Arthrocnemum macrostachyum and Salicornia ramosissima populations, the stigma protrudes from the perianth when the anthers have shed their pollen. In the Sarcocornia fruticosa and Sarcocornia perennis populations, anthesis starts with the extrusion of one anther, but in the most common pattern the stigma protrudes from the perianth while the anthers are shedding their pollen. All species studied were morphologically protandrous, but only in one population of Arthrocnemum macrostachyum was the number of germinated pollen grains on the stigma significantly greater when the stigma was exserted than in the previous phases.     

Gynoecium structure and extra-gynoecial pollen-tube growth in an apocarpous species, <Emphasis Type="Italic">Sagittaria trifolia</Emphasis> (Alismataceae)

Apocarpy is regarded as an original feature obtained during the evolution of angiosperms. Compared with syncarpous plants, apocarpous plants have some adaptive disadvantages in apocarpous plants, for example, the number of offspring is lower under conditions of uneven pollen-tube distribution. However, in some apocarpous species, extra-gynoecial pollen-tube growth (EGPG) may remedy this disadvantage. We conducted micro-observations and field studies of Sagittaria trifolia, to investigate the gynoecium structure and the pathway of pollen-tube growth in the entire gynoecium. In a single-carpel pollination experiment, we found that the extra-gynoecial pollen tubes from a carpel of S. trifolia were able to fertilize approximately 13 carpels. Simulated EGPG in the entire gynoecium of S. trifolia revealed that its effect on the seed set could be divided into two stages: stage of low/high-level stigmas pollination, in which the cutoff point was about 0.1. The seed set would be markedly improved during the low-level stigmas pollination stage by EGPG when the maximum distance of extra-gynoecial pollen tubes could span three carpels, as in the present experiment. Our simulation also showed that the high pollen load could enhance the effect of EGPG on the seed set, and if the number of germinating pollen is triple the carpel number in the gynoecium, a 100% seed set rate would be obtained when approximately 50% of the stigmas are pollinated.     

Holocentric chromosome behaviour inCuscuta (Cuscutaceae)

The present study demonstrates thatCuscuta babylonica Choisy has holocentric chromosomes. Evidence for this phenomenon comes from three different observations. (1) Mitosis: During metaphase and anaphase the sister-chromatids are situated parallel to the equatorial plane with no sign of localized kinetochore activity. (2) Inverted meiosis in microsporocytes. (3) X-rayed microsporocytes, in which the numerous chromosome fragments do not show any lagging or formation of micronuclei. We assume that only one out of the three subgenera inCuscuta, namely subg.Cuscuta, has holocentric chromosomes, while the two other subgenera have monocentric chromosomes.     

桦木科植物花柱适应风媒传粉的特征

植物体为适应自己的传粉系统, 表现出高度的适应特征。风媒花植物为适应风传播花粉, 要形成特殊的结构, 以扩大接受花粉粒的面积。利用扫描电镜观察了桦木科(Betulaceae)6属18种植物花柱的形态及花粉粒在花柱上的萌发过程, 探讨了桦木科植物花柱适应风媒传粉的特征。结果表明, 桦木科植物的二心皮(铁木、云南鹅耳枥稀为三心皮)雌蕊具柱状花柱, 柱头不发达, 花柱表皮细胞长条状, 纵向排列紧密。传粉时, 花柱表皮细胞能执行柱头的功能, 接受花粉粒, 为花粉粒萌发提供场所和萌发条件。桦木科植物花柱有2种类型: 一种是花柱表皮细胞能形成乳突, 花粉管经乳突细胞进入花柱; 另一种是花柱表皮细胞不形成乳突, 花粉管经过花柱表皮细胞或胞间隙进入花柱。无论花柱表皮细胞是否形成乳突, 乳突的形态、大小以及花粉管和乳突的结合方式等在族间、属间、属内种间存在差异。与基部被子植物相比, 桦木科植物的花柱呈现适应风媒传粉的进化特征。桦木科植物花柱表皮细胞形成的乳突与基部被子植物柱头乳突功能相同, 是桦木科植物风媒传粉的适应策略。     

SD-RLK28 positively regulates pollen hydration on stigmas as a PCP-Bβ receptor in Arabidopsis thaliana

Pollen hydration on dry stigmas is strictly regulated by pollen–stigma interactions in Brassicaceae. Although several related molecular events have been described, the molecular mechanism underlying pollen hydration remains elusive. Multiple B-class pollen coat proteins(PCP-Bs) are involved in pollen hydration. Here, by analyzing the interactions of two PCP-Bs with three Arabidopsis thaliana stigmas strongly expressing S-domain receptor kinase(SD-RLK), we determined that SD-RLK28 directly intera...     

Stigma morphology in distylous and non-heterostylous species ofVillarsia (Menyanthaceae)

Stigma morphology was examined with the SEM in 14 of the 16 species ofVillarsia. In nine of the ten distylous species studied, stigmas of the floral morphs were strongly dimorphic in length, shape, configuration of the receptive surface, and in the size and density of their papillae. Thrum stigmas ofVillarsia, in contrast to those of most other distylous species, are not simply smaller versions of the conspecific pin stigmas, but generally exhibit an array of morph-specific characters. Thrum stigma lobes may be broader than those of pins, they may have undulate margins, lobes subdivided into secondary lobes, papillae more extensively distributed than in pins, and various combinations of these traits occur. The traits that distinguish thrum from pin stigmas achieve an increase in the receptive area and may enhance more efficient pollen capture by the shorter and less accessible thrum stigmas. The morphogenesis of the stigma shape dimorphism appears to involve processes more complex than inhibition of elongation in thrum styles. InVillarsia, the stigma dimorphisms are species-specific. No correlations were found between morphologies of the stigma and the different breeding systems in distylous species. Stigmas of the four non-heterostylous species examined resemble the thrum stigma type found in most distylousVillarsia species.     

Lack of Control by Early Pistillate Ethylene of the Accelerated Wilting of Petunia hybrida Flowers

Well before pollen tube penetration, ethylene has begun to disseminate from pollinated styles of Petunia hybrida flowers. Previous stigmatic application of aminoethoxyvinylglycine (AVG) completely prevented this ethylene synthesis, indicating that the endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) in pollen is not readily converted on the stigma. Compared to other flower parts, the capacity of the ethylene forming enzyme was largest in the stigma. When applied to the stigma, ACC caused ethylene synthesis, but did not accelerate wilting, unless high concentrations (20 nanomols) were used. Upon pollination or stigma wounding, the early ethylene evolved exclusively from the gynoecium, much later followed by the synthesis of corolla ethylene. Employing wideneck Erlenmeyer flasks, the competitive inhibitor of ethylene action, norbornadiene, was applied to entire flowers in situ, with delaying effects on wound-induced wilting. In contrast, norbornadiene treatment of styles alone, using capillaries, could not postpone wilting. Pollination with foreign pollen species did not lead to accelerated corolla wilting, notwithstanding considerable synthesis of ethylene during the first 5 hours. In situ treatment of the stigma with AVG considerably delayed wound- and pollination-induced wilting. Removal of the entire AVG-treated style 6 hours after stigma wounding still allowed for the postponement of the accelerated wilting, even at very low concentrations of AVG. It is concluded that early stylar ethylene does not play a role in the acceleration of wilting but that, much later, corolla ethylene does, induced by a mobile wilting factor from the stigma, which is ACC.