Former diversity of Ephedra (Gnetales): evidence from Early Cretaceous seeds from Portugal and North America

BACKGROUND AND AIMS: The extant species of the seed plant group Gnetales (Ephedra, Gnetum and Welwitschia) have been considered a remnant of a much greater, now extinct, diversity due to the pronounced differences in form and ecology among the genera. Until recently, this hypothesis has not been supported by evidence from the fossil record. This paper adds to the expanding information on Gnetales from the Early Cretaceous and describes coalified seeds from Barremian-Albian localities in Portugal and USA. METHODS: The fossils were extracted from sediment samples by sieving in water. Adhering mineral matrix was removed by chemical treatment. Seeds were investigated using light and scanning electron microscopy. Morphology and anatomy of the seeds were documented and compared with those of extant species. KEY RESULTS: The fossils share characters with extant Ephedra, for example papillae on the inner surface of the seed envelope and in situ polyplicate pollen grains that shed the exine during germination. They differ from extant Ephedra seeds in morphological and anatomical details as well as in their smaller size. Two new species of Ephedra are described together with one species assigned to a new genus of Gnetales. Other Ephedra-like seeds, for which pollen and critical morphological details are currently unknown, are also present in the samples. CONCLUSIONS: These Cretaceous seeds document that key reproductive characters and pollen germination processes have remained unchanged within Ephedra for about 120 million years or more. There is sufficient variety in details of morphology to suggest that a diversity of Ephedra and Ephedra-like species were present in the Early Cretaceous flora. Their presence in Portugal and eastern North America indicates that they were widespread on the Laurasian continent. The fossil seeds are similar to seeds of Erdtmanithecales and this supports the previously suggested relationship between Erdtmanithecales and Gnetales.     

From near extinction to diversification by means of a shift in pollination mechanism in the gymnosperm relict Ephedra (Ephedraceae,Gnetales)

Pollination in gymnosperms is usually accomplished by means of wind, but some groups are insect‐pollinated. We show that wind and insect pollination occur in the morphologically uniform genus Ephedra (Gnetales). Based on field experiments over several years, we demonstrate distinct differences between two Ephedra species that grow in sympatry in Greece in pollen dispersal and clump formation, insect visitations and embryo formation when insects are denied access to cones. Ephedra distachya, nested in the core clade of Ephedra, is anemophilous, which is probably the prevailing state in Ephedra. Ephedra foeminea, sister to the remaining species of the genus, is entomophilous and pollinated by a range of diurnal and nocturnal insects. The generalist entomophilous system of E. foeminea, with distinct but infrequent insect visitations, is in many respects similar to that reported for Gnetum and Welwitschia and appears ancestral in Gnetales. The Ephedra lineage is well documented already from the Early Cretaceous, but the diversity declined dramatically during the Late Cretaceous, possibly to near extinction around the Cretaceous–Palaeogene boundary. The clade imbalance between insect‐ and wind‐pollinated lineages is larger than expected by chance and the shift in pollination mode may explain why Ephedra escaped extinction and began to diversify again.     

Polarity, aperture condition and germination in pollen grains ofEphedra (Gnetales)

Pollen grain polarity, aperture condition and pollen tube formation were examined inEphedra americana, E. foliata, E. rupestris, E. distachya, andE. fragilis using LM, SEM and TEM. In the characteristic oblate pollen, as seen in situ in the tetrad configuration, the polar axis is the minor one and the equatorial plane runs between the two narrow ends of the microspore. The intine is thick in fresh fixed mature pollen but we have seen no indication of regions having an exceptionally thick intine that could be considered associated with an aperture or apertures. About three minutes after transferring fresh pollen to the germinating medium the ridged exine splits and twists away from the intine and its enclosed protoplast. The shed exine spreads out and curls into a scroll-like configuration that is as distinctive as that of the pollen shape had been but now having the ridges and valleys perpendicular to the long axis. The pollen tube develops, in our experience with more than a hundred germinating pollen grains, near one of the narrow tips of the pollen grain's equatorial plane. The location of the pollen tube initiation probably is related to the position of the tube cell nucleus. The pollen tube starts to grow about one hour after the exine was shed. The pollen tube emerges close to the narrow end (equator) of the gametophyte. This end emerged first as the exine is shed and is opposite to the prothallial cells. The stout pollen tube is c. 10µm in diameter grown in vitro on agar. In our germination medium the stout tube continued to elongate for about 24 hours reaching a length of c. 100 µm. With respect to exine morphology the aperture condition could be considered as inaperturate. The pollen tube, however, is formed in a germination area near one end of the exineless gametophyte.     

Beschorneria yuccoides and Asimina triloba (L.) Dun: Examples for proximal polar germinating pollen in angiosperms

Beschorneria yuccoides (Agavaceae) microspores are arranged mostly in planar tetrads. Later on, the pollen grains of the tetrad usually fall apart, but sometimes remain loosely connected by ektexine elements. The ektexine consists of a tectum, of short columellae, and of a thin, discontinuous foot layer. An endexine is absent. The bilayered intine is without any additional thickening that would usually indicate an aperture region. From this point of view the pollen grain might be considered as omniaperturate. The pollen ornamentation is reticulate with wide lumina and robust, smooth muri.

The pollen grains show an indistinct sulcus characterised by a loose reticulate ornamentation. The sulcus is not exactly at the distal pole, but shifted towards the equator. No pollen tubes are formed regularly at the sulcus. Instead, pollen tubes are normally formed at the proximal pollen face. The proximal area, indicating a large germination field, is morphologically and functionally clearly an aperture (a germination zone); however, it does not represent a sulcus. The proximal face of all pollen grains appears as ornamented, with some exine lumps.

Asimina triloba (Annonaceae) pollen is shed in permanent planar or decussate tetrads. The distal sides are microreticulate to foveolate, and do not show an aperture; the psilate proximal sides are the germination areas of A. triloba.

The presence of apertures placed at the proximal pole was reported for distinct taxa of several angiosperm families. For Drosera, Dionaea (Droseraceae) and most probably for the diaperturate Cuphea species (Lythraceae) the existence of polar germination areas can be excluded. However, in some Annonaceae taxa with permanent tetrads (Annona cherimola, Asimina triloba) a situation similar to Beschorneria might be present, and indeed a proximal polar pollen tube is formed. Beschorneria yuccoides, Annona cherimola and Asimina triloba are unequivocal examples of angiosperm pollen with an exactly proximal aperture (germination area).     

The Gnetales: Recent insights on their morphology,reproductive biology,chromosome numbers,biogeography, and divergence times

Ephedra, Gnetum, and Welwitschia constitute the gymnosperm order Gnetales of still unclear phylogenetic relationships within seed plants. Here we review progress over the past 10 years in our understanding of their species diversity, morphology, reproductive biology, chromosome numbers, and genome sizes, highlighting the unevenness in the sampling of species even for traits that can be studied in preserved material, such as pollen morphology. We include distribution maps and original illustrations of key features, and specify which species groups or geographic areas are undersampled.

     

Air pollution effects on structure,proteins and flavonoids in pollen grains of Thuja orientalis L. (Cupressaceae)

Increase in the levels of air pollution due to the increase in industrial and agricultural technology has prompted investigation of mechanisms that contribute to air pollution tolerance in plants. Pollen grains of Thuja orientalis were collected from controlled (less polluted) and polluted areas (mainly SO₂, NO₂, CO, HC and APM). Thuja pollen is considered inaperturate and granulate. The exine is shed during rehydration, leaving the male gametophyte naked. The pollen grains collected from polluted areas are smaller and more fragile compared to control ones. The exine splitting is faster and higher in polluted pollen grains. SDS–PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) pattern do not show significant differences in polluted pollen than those in the control group. HPLC (high pressure liquid chromatography) analysis demonstrates that air pollution induces flavonoids accumulation to significantly higher levels in polluted pollen than in controlled ones. These observations suggest that plants try to respond suitably by adjusting their metabolism so that minimum damage is done due to air pollutants. Their protective responses may include an increase in antioxidant enzymes and metabolites and induction of protection-related secondary metabolite genes especially flavonoids.     

Double fertilization in Gnetum gnemon (Gnetaceae): its bearing on the evolution of sexual reproduction within the Gnetales and the anthophyte clade

The fertilization process in Gnetum is critical to our understanding of the evolution of sexual reproduction within the Gnetales, a monophyletic group of nonfiowering seed plants that are the closest living relatives to flowering plants. Although much is known about the fertilization process in Ephedra, which is basal within the Gnetales, little is known about sexual reproduction in the derived sister groups Gnetum and Welwitschia. Ovules of Gnetum gnemon were collected at various stages after hand pollination and processed for light, fluorescence, and electron microscopy. Approximately 5 d after pollination, pollen tubes reach sexually mature female gametophytes, which are coenocytic. At that time, a binucleate sperm cell is found within each pollen tube. Within 7 d of pollination, double fertilization events occur when each of two sperm nuclei released from a pollen tube fuses with a separate, undifferentiated female nucleus within the free nuclear female gametophyte, which lacks differentiated egg cells. The products of double fertilization are two viable zygotes; endosperm is not formed. The lack of differentiated egg cells in Gnetum gnemon is unparalleled among land plants and the documentation of a regularly occurring process of double fertilization is congruent with the hypothesis that a rudimentary process of double fertilization evolved in a common ancestor of angiosperms and Gnetales.     

A fossil‐calibrated relaxed clock for Ephedra indicates an Oligocene age for the divergence of Asian and New World clades and Miocene dispersal into South America

Abstract Ephedra comprises approximately 50 species, which are roughly equally distributed between the Old and New World deserts, but not in the intervening regions (amphitropical range). Great heterogeneity in the substitution rates of Gnetales (Ephedra, Gnetum, and Welwitschia) has made it difficult to infer the ages of the major divergence events in Ephedra, such as the timing of the Beringian disjunction in the genus and the entry into South America. Here, we use data from as many Gnetales species and genes as available from GenBank and from a recent study to investigate the timing of the major divergence events. Because of the tradeoff between the amount of missing data and taxon/gene sampling, we reduced the initial matrix of 265 accessions and 12 loci to 95 accessions and 10 loci, and further to 42 species (and 7736 aligned nucleotides) to achieve stationary distributions in the Bayesian molecular clock runs. Results from a relaxed clock with an uncorrelated rates model and fossil‐based calibration reveal that New World species are monophyletic and diverged from their mostly Asian sister clade some 30 mya, fitting with many other Beringian disjunctions. The split between the single North American and the single South American clade occurred approximately 25 mya, well before the closure of the Panamanian Isthmus. Overall, the biogeographic history of Ephedra appears dominated by long‐distance dispersal, but finer‐scale studies are needed to test this hypothesis.     

Morphology and lateral germination of pollen in Ginkgo biloba and their implications in evolution

The four-celled mature pollen of Ginkgo biloba L. just shed from the microsporangia, was round in shape and contained a very large germination area with monosulcate sulcus walled with two semicircular and nearly perpendicular parts of exine, and the pollen was coated with a compact irregular striations and spinules in parts other than the germination area. The pollen became boatshaped and was bilaterally symmetrical when it was in air or under cold storage, but rapidly rounded off in cultural solution. Studies on early morphogenesis of the male gametophyte in vitro and in vivo revealed that the tube nucleus turned a certain degree of angle, and was followed by lateral germination, while the non-active pollen maintained its round shape. The results suggest that the internal feature of the pollen tube could be a determinant factor contributing to the expression of the characteristic events of lateral germination of the male gametophyte in G. biloba. Furthermore, this biologically consistent character, together with the characteristic pollen morphology, is quite different from those of other groups in gymnosperms such as Cycas and Pinus. This might be a critical characterrelevant to the systematic position of G. biloba.     

Pollen evolution and systematics inAnnonaceae with special reference to the disulcate Australian endemic genera

All genera ofAnnonaceae endemic in Australia (Ancana, Fitzalania, Haplostichanthus) show almost exactly the same type of disulcate (disulculate) pollen with intact exine extending over the sulci. Tetrad stages inHaplostichanthus andAncana reveal a latudinal subequatiorial orientation of the two sulci at the proximal hemisphere. Sometimes they fuse into a ±zonosulcate aperture.Fissistigma pollen grains are ±globose and have a flattened pole with a central elevation and a concentric groove, covered by a somewhat reduced exine. This palynological characters give further support for separating the generaAncana andFissistigma. Germination was observed inHaplostichanthus where the pollen tube emerges at one of the two sulci and inFissistigma where the flattened part breaks up during germination. The aperture types described here are obviously transitional stages between aperturate and inaperturate pollen grains and are discussed in regard to pollen evolution.     

Pollen morphology in the family Asphodelaceae (Asphodeleae,Kniphofieae)

Pollen of 72 species from 7 genera of the Asphodelaceae was studied by means of LM and SEM in order to investigate their taxonomic relationships. The pollen of all the genera investigated is heteropolar, monosulcate, non-operculate and differs in size, form and exine ornamentation. The species of Eremurus are homogenous palynologically and studies of this genus do not support the segregation of the genera Henningia, Selonia, and Ammolirion. The pollen of Bulbinopsis mainly differs from the pollen of Bulbine species in having a perforate-areolate exine surface (B. bulbosa) but the genera are close in their other palynological features. The genera Asphodelus and Asphodeline have the largest, almost spheroidal grains with the thickest exine but differ in details of exine ornamentation. The species of Kniphofia are very close palynologically.     

Ephedroid fossil pollen from the Lower Cretaceous (Upper Albian) of Hokkaido,Japan

This paper presents eight ephedroid palynomorphs from the Lower Cretaceous (Upper Albian) of Hokkaido, Japan. The ephedroid palynomorphs are ellipsoid and polyplicate pollen grains that show a wide range of variation in pollen size, shape, and plication. These ephedroid palynomorphs suggest a wide range of diversity in Gnetales at mid- or high-paleolatitude in the eastern side of Lauresia during the Upper Albian.     

银杏成熟花粉的形态、侧向萌发及其意义

刚从小孢子囊中散发出的银杏Ginkgo biloba L．成熟花粉为圆球形,具有巨大的单一萌发区。萌发区由两部分半圆形的、几近垂直的外壁围成。除萌发区外,花粉的外壁被有较致密的不规则的纹理和小刺。当花粉在空中或冷储条件下滞留一段时间后,即转变为两侧对称的船形,但在培养液中又很快转变为近圆球形。离体和活体实验都表明,银杏雄配子体萌发初期,管核都转过了一定的角度,并继而发生侧向萌发,但没有活性的花粉除变为圆球形外无其他变化。这些表明,花粉管的内部特征决定了银杏雄配子体侧向萌发的典型特征。这一稳定的生物学特性,加上银杏花粉的特殊形态特征,与苏铁属、松属等有明显的区别。这可能是银杏特殊系统位置的一个关键特征。     

THE ULTRASTRUCTURE OF SAHNIA POLLEN (PENTOXYLALES)

The micromorphology and fine structure of in situ pentoxylalean pollen are described from the holotype of Sahnia laxiphora Drinnan and Chambers 1985 collected from the Lower Cretaceous (Valanginian-Aptian) of Victoria, southeastern Australia. Pollen grains are ovoid, monosulcate, and relatively small, averaging 26 μm in length. Exine ornamentation is psilate. The sporoderm is two-parted with the sexine staining lightly throughout and approximately six times the thickness of the more darkly staining nexine. The exine over the sulcus is typically strongly invaginated, and may or may not include an extremely thin sexine layer. The outer part of the sexine is homogeneous, while the inner part is composed of relatively large granules separated by irregular lacunae of various sizes; lacunae are most pronounced at the sexinenexine interface. Faint lamellae characterize the nexine in both apertural and nonapertural regions. Granular orbicules are often associated with the exine surfaces and also occur appressed to pollen sac walls along with lamellated tapetal membranes. Sporoderm ultrastructure is compared to that of nonsaccate pollen of other groups, and particularly to pollen of Bennettitales, Gnetales, angiosperms, and similar plants, to which the Pentoxylales have been thought to be closely related. Although Sahnia laxiphora pollen is not identical to that of any of these taxa, the strongest similarity is with pollen of Bennettitales.     

Pollen Morphology of the Periplocaceae from Madagascar

The pollen morphology of eleven (Baroniella Constantin & Galland, Baseonema Schltr. & Rendle, Camptocarpus Decne., Cryptolepis R. Br., Cryptostegia R. Br., Gonocrypta Baill., Harpanema Decne., Ischnolepis Jum. & H. Perrier, Menabea Baill., Pentopetia Decne., and Tanulepis Balf. f.) of the 13 genera of the Periplocaceae from Madagascar have been examined using light-, scanning-, and transmission electron microscopy. All of the genera are characterized by pollen grains arranged in tetrads. The arrangement of the grains may be rhomboidal, decussate or tetragonal. The 4–6 pores present are restricted to the junction area of adjacent grains. Cryptostegia differs from the other genera in that the arrangement of pollen grains is only decussate. In Menabea the tetrads are united into a pollinium. The exine is smooth and consists of a distal stratum (tectum), subtended by a granular stratum consisting of granules of unequal size. Towards the base larger granules are present. In Camptocarpus, Harpanema and Tanulepis the exine is stratified into a distal stratum (tectum), a thin granular stratum and an almost continuous basal stratum (foot layer). The inline is well developed. The pollen grains of tetrads are connected by wall bridges (cross-wall cohesion). The internal walls in Camptocarpus, Harpanema and Tanulepis differ from the other genera in the absence of a tectum. The pollen morphology of the taxa investigated is very similar and of little value for distinguishing the species and genera investigated. The distinctive difference in exine structure between the above mentioned three genera and other genera investigated emphasizes the importance of exine ultrastructure in the Periplocaceae.     

Pollination biology of Gnetum (Gnetaceae) in a LOWLAND MIXED DIPTEROCARP forest in Sarawak

Pollination biology of two Gnetum species was studied in a lowland mixed dipterocarp forest in Sarawak, Malaysia. A dioecious shrub species, G. gnemon var. tenerum, flowered in the evening and its strobili emitted a stinking odor. Pollination droplets were secreted from ovules on female strobili and from sterile ovules on male strobili in the evening. These strobili were visited by nectar-seeking moths of Pyralidae and Geometridae. Sticky pollen was attached on proboscides and antennae of these moths. A dioecious woody climber, G. cuspidatum, had cauline strobili on woody stems near the forest floor. Its male strobili emitted a funguslike odor in the evening and secreted nectar on collars that subtended “flower” rings. The strobili were visited by small flies of Lauxaniidae (Diptera), onto the antennae and legs of which pollen was attached. Evaporation of the exposed droplet/nectar of these Gnetum flowers seemed to be minimized by nocturnal flowering near the tropical rain forest floor. Another floral-structural constraint of gymnosperms, i.e., lack of showy petals, was compensated for by floral fragrancy. The prevalence of entomophily in the Gnetales (i.e., Gnetum, Ephedra, and Welwitschia) may suggest that unspecialized insect pollination originated before the divergence between the Gnetales and angiosperms.     

Ultrastruktur und systematische Bedeutung des Pollens beiBocageopsis,Ephedranthus, Malmea undUnonopsis (Annonaceae)

The four genera investigated show solitary sulcate pollen grains. The structure and sculpture of the sporoderm is very similar inBocageopsis andUnonopsis, and supports the close relationship of both genera; their flower morphology also is very similar. In contrast, the sporoderm ofEphedranthus, and especially that ofMalmea, is different in some characters and suggests more remote relationships; this is also confirmed by differences in flower morphology. Within this group of genera a distinct exine progression can be recognized from non-columellate (Malmea) to granular (Unonopsis guatterioides), further to somewhat irregularly (Bocageopsis, someUnonopsis species), and finally to very regularly columellate (Ephedranthus). The sculpture of the tectum varies from a reticulum with large lumina (Malmea) to one with small performations (Ephedranthus). Within theAnnonaceae the genusMalmea is among the most primitive in respect to pollen structure. The sulcus of the four genera is very large and runs over 1/3 of the pollen grain. It is characterized by a reduction of the exine and a bulgy thickening of the intine.

     

Correlation between pollen morphology and pollination mechanisms in the Hydrocharitaceae

The pollen morphology of 11 genera and 11 species of the Hydrocharitaceae and one species of the Najadaceae was studied using scanning and transmission electron microscopies, and the exine structures and sculptures are discussed in relation to pollination mechanisms and the molecular phylogeny. The pollen grains of the Hydrocharitaceae are spherical, inaperturate, and form monads or tetrads, while those of the Najadaceae are elliptical, inaperturate, and form monads. The entomophilous genera Egeria, Blyxa, Ottelia, Stratiotes, and Hydrocharis share pollen grains that have projections like spines or bacula. The anemophilous genus Limnobium has reticulate pollen grains. The hypohydrophilous genera Thalassia and Najas are characterized by pollen grains with reduced exine structures. The pollen-epihydrophilous genera Elodea and Hydrilla have tightly arranged small spinous pollen grains, and the male flower-epihydrophilous genera Enhalus and Vallisneria have reduced reticulate or gemmate exines. Character state reconstruction of the exine structures and sculptures using a molecular phylogenetic tree suggests that variation in the exine is generally correlated with the pollination mechanism; the selective pressures acting on the pollination mechanisms have reduced the exine structure in hypohydrophilous plants and resulted in various exine sculptures that are adapted to the different pollination mechanisms in entomophilous, anemophilous, and pollen-epihydrophilous plants.     

Aerodynamics of Ephedra trifurca

Computer simulations are used to predict the behavior of pollen grains with different physical properties within the acceleration field created around the ovules of the gymnosperm Ephedra trifurca. A modelling procedure is given that (1) calculates the number of pollen grains captured by an ovule's pollination-droplet and (2) gives a correlation between pollination efficiency and the physical properties (= mass, size) of different types of pollen. Based on this procedure, the number of Ephedra pollen grains captured by micropyles can be less than the number captured from other species. However, the mass and size of Ephedra pollen grains appear to coincide with those predicted to yield a local maximum of pollination efficiency, i.e. slightly larger or smaller values of either mass or size would decrease the probability of capture. In addition, the properties of Ephedra pollen grains operate synergistically in the aerodynamic environment around ovules and are focused to collide with pollination-droplets. By analogy, the properties of Ephedra pollen coincide with those predicted for a localized adaptive peak. The physical properties of pollen grain types other than E. trifurca that can maximize pollen capture are not generally represented in the aerobiology of Ephedra during the pollination season. Therefore, the phenology of pollen release, community taxonomic-composition, and the physics of particle capture play collectively important roles in the reproductive success of Ephedra trifurca.     

Aperture types withinSapranthus andPolyalthia (Annonaceae)

Within the large palaeotropical genusPolyalthia so far five different pollen types are described (two monosulcate types, two disulcate types, and a single omniaperturate type). One of the disulcatePolyalthia types is similar to the disulcate neotropical genusSapranthus. All the newly described types differ predominantly not in exine but rather in intine characters. From this it can be concluded that seemingly inaperturate grains are disculcate in function, generally with two clearly recognizable germination zones, or omniaperturate, with the whole sporoderm capable of germination. The functions of the intine layers within the germination zones and within the nongerminating areas are very different and cooperate during the first germination steps in bursting the exine. It is difficult clearly to define the germination zones of pollen grains if the exine exhibits no modifications. Based on our and other results a more precise definition is suggested.