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.

Most gymnosperms are wind-pollinated, but some are insect-pollinated, and in Ephedra (Gnetales), both wind pollination and insect pollination occur. Little is, however, known about mechanisms and evolution of pollination syndromes in gymnosperms. Based on four seasons of field studies, we show an unexpected correlation between pollination and the phases of the moon in one of our studied species, Ephedra foeminea. It is pollinated by dipterans and lepidopterans, most of them nocturnal, and its pollination coincides with the full moon of July. This may be adaptive in two ways. Many nocturnal insects navigate using the moon. Further, the spectacular reflection of the full-moonlight in the pollination drops is the only apparent means of nocturnal attraction of insects in these plants. In the sympatric but wind-pollinated Ephedra distachya, pollination is not correlated to the full moon but occurs at approximately the same dates every year. The lunar correlation has probably been lost in most species of Ephedra subsequent an evolutionary shift to wind pollination in the clade. When the services of insects are no longer needed for successful pollination, the adaptive value of correlating pollination with the full moon is lost, and conceivably also the trait.

The fast Ut and dense ultrastructure of E. foeminea pollen are consistent with functional traits that distinguish entomophilous species from anemophilous species. In anemophilous Ephedra species, ovulate organs create an aerodynamic microenvironment that directs airborne pollen to the pollination drops. In E. foeminea, no such microenvironment is created. Ephedroid palynomorphs from the Cretaceous share the ultrastructural characteristics of E. foeminea, and at least some may, therefore, have been produced by insect-pollinated plants.

The Ephedra lineage can be traced at least to the Early Cretaceous. Its characteristically polyplicate pollen is well-represented in the fossil record and is frequently used as an indicator of paleoclimate. However, despite previous efforts, knowledge about variation and evolution of ephedroid pollen traits is poor. Here, we document pollen morphology of nearly all extant species of Ephedra, using a combination of scanning electron microscopy (SEM) and light microscopy (LM), and reconstruct ancestral states of key pollen traits. Our results indicate that the ancestral Ephedra pollen type has numerous plicae interspaced by unbranched pseudosulci, while the derived pollen type has branched pseudosulci and (generally) fewer plicae. The derived type is inferred to have evolved independently twice, once along the North American stem branch and once along the Asian stem branch. Pollen of the ancestral type is common in Mesozoic fossil records, especially from the Early Cretaceous, but it is less commonly reported from the Cenozoic. The earliest documentation of the derived pollen type is from the latest Cretaceous, after which it increases strongly in abundance during the Paleogene. The results of the present study have implications for the age of crown group Ephedra as well as for understanding evolution of pollination syndromes in the genus.

International audienceSteppe vegetation represents a key marker of past Asian aridification and is associated with monsoonal intensification. Littleis, however, known about the origin of this pre-Oligocene vegetation, its specific composition and how it changed over timeand responded to climatic variations. Here, we describe the morphological characters of Ephedraceae pollen in Eocene strataof the Xining Basin and compare the pollen composition with the palynological composition of Late Cretaceous andPaleocene deposits of the Xining Basin and the Quaternary deposits of the Qaidam Basin. We find that the Late Cretaceoussteppe was dominated by Gnetaceaepollenites; in the transition from the Cretaceous to the Paleocene, Gnetaceaepollenitesbecame extinct and Ephedripites subgenus Ephedripites dominated the flora with rare occurrences of Ephedripites subgen.Distachyapites; the middle to late Eocene presents a strong increase of Ephedripites subgen. Distachyapites; and the Quaternary/Recent is marked by a significantly lower diversity of Ephedraceae (and Nitrariaceae) compared to the Eocene. In themodern landscape of China, only a fraction of the Paleogene species diversity of Ephedraceae remains and we propose thatthese alterations in Ephedreaceae composition occurred in response to the climatic changes at least since the Eocene. Inparticular, the strong Eocene monsoons that enhanced the continental aridification may have played an important role inthe evolution of Ephedripites subgen. Distachyapites triggering an evolutionary shift to wind-pollination in this group.Conceivably, the Ephedraceae/Nitrariaceae dominated steppe ended during the Eocene/Oligocene climatic cooling andaridification, which favoured other plant taxa