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.

Cretaceous chloranthoids: early prominence, extinct diversity and missing links.

BACKGROUND: The Chloranthaceae comprise four extant genera (Hedyosmum, Ascarina, Chloranthus and Sarcandra), all with simple flowers. Molecular phylogenetics indicates that the Chloranthaceae diverged very early in angiosperm evolution, although how they are related to eudicots, magnoliids, monocots and Ceratophyllum is uncertain. Fossil pollen similar to that of Ascarina and Hedyosmum has long been recognized in the Early Cretaceous, but over the last four decades evidence of extinct Chloranthaceae based on other types of fossils has expanded dramatically and contributes significantly to understanding the evolution of the family. SCOPE: Studies of fossils from the Cretaceous, especially mesofossils of Early Cretaceous age from Portugal and eastern North America, recognized diverse flowers, fruits, seeds, staminate inflorescences and stamens of extinct chloranthoids. These early chloranthoids include forms related to extant Hedyosmum and also to the Ascarina, Chloranthus and Sarcandra clade. In the Late Cretaceous there are several occurrences of distinctive fossil androecia related to extant Chloranthus. The rich and still expanding Cretaceous record of Chloranthaceae contrasts with a very sparse Cenozoic record, emphasizing that the four extant genera are likely to be relictual, although speciation within the genera might have occurred in relatively recent times. In this study, we describe three new genera of Early Cretaceous chloranthoids and summarize current knowledge on the extinct diversity of the group. CONCLUSIONS: The evolutionary lineage that includes extant Chloranthaceae is diverse and abundantly represented in Early Cretaceous mesofossil floras that provide some of the earliest evidence of angiosperm reproductive structures. Extinct chloranthoids, some of which are clearly in the Chloranthaceae crown group, fill some of the morphological gaps that currently separate the extant genera, help to illuminate how some of the unusual features of extant Chloranthaceae evolved and suggest that Chloranthaceae are of disproportionate importance for a more refined understanding of ecology and phylogeny of early angiosperm diversification.

Mongolitria: A new Early Cretaceous three-valved seed from Northeast Asia.

PREMISE: Fossil seeds recovered from the Early Cretaceous of Mongolia and Inner Mongolia, China, are described and assigned to Mongolitria gen. nov., a new genus of gymnosperm seed. METHODS: Abundant lignitized seeds along with compression specimens isolated from the matrix were studied using a combination of scanning electron microscopy, anatomical sectioning, light microscopy, synchrotron radiation X-ray microtomography, and cuticle preparations. A single permineralized seed was examined by light microscopy of cellulose acetate peels and X-ray microtomography. RESULTS: Two species are recognized, Mongolitria friisae sp. nov. and Mongolitria exesum sp. nov. Both seeds are orthotropous with a short apical micropyle and a small, basal, circular attachment scar. The thick sclerenchymatous integument has a consistently three-parted organization and about 20 conspicuous longitudinal ribs on the surface. Mongolitria exesum differs from M. friisae primarily in its much larger size and thicker seed coat, which also preserves clear evidence of insect damage. CONCLUSIONS: Mongolitria is similar to other fossil seeds that have been assigned to Cycadales, but displays a unique combination of characters not found in any living or extinct cycadaceous plant, leaving its higher-level systematic affinities uncertain. Germination apparently involved splitting of the integument into three valves. Mongolitria was prominent among the plant parts accumulating in peat swamps in eastern Asia during the Early Cretaceous.

Estimates of late Early Cretaceous atmospheric CO2 from Mongolia based on stomatal and isotopic analysis of Pseudotorellia.

PREMISE: The Aptian-Albian (121.4-100.5 Ma) was a greenhouse period with global temperatures estimated as 10-15°C warmer than pre-industrial conditions, so it is surprising that the most reliable CO2 estimates from this time are <1400 ppm. This low CO2 during a warm period implies a very high Earth-system sensitivity in the range of 6 to 9°C per CO2 doubling between the Aptian-Albian and today. METHODS: We applied a well-vetted paleo-CO2 proxy based on leaf gas-exchange principles (Franks model) to two Pseudotorellia species from three stratigraphically similar samples at the Tevshiin Govi lignite mine in central Mongolia (~119.7-100.5 Ma). RESULTS: Our median estimated CO2 concentration from the three respective samples was 2132, 2405, and 2770 ppm. The primary reason for the high estimated CO2 but with relatively large uncertainties is the very low stomatal density in both species, where small variations propagate to large changes in estimated CO2. Indeed, we found that at least 15 leaves are required before the aggregate estimated CO2 approaches that of the full data set. CONCLUSIONS: Our three CO2 estimates all exceeded 2000 ppm, translating to an Earth-system sensitivity (~3-5°C/CO2 doubling) that is more in keeping with the current understanding of the long-term climate system. Because of our large sample size, the directly measured inputs did not contribute much to the overall uncertainty in estimated CO2; instead, the inferred inputs were responsible for most of the overall uncertainty and thus should be scrutinized for their value choices.

Reconstruction of an enigmatic Pennsylvanian cone reveals a relationship to Sphenophyllales.

PREMISE: We studied the 3D morphology of a small, well-preserved cone from the Pennsylvanian Mazon Creek Lagerstätte to characterize its structure and determine its systematic affinity. Previously tentatively assigned to the enigmatic Tetraphyllostrobus, we show that it differs in key respects from that genus as described. METHODS: We systematically compared the new fossil with relevant Paleozoic cone genera and employed advanced imaging techniques, including scanning electron microscopy, Airyscan confocal super-resolution microscopy, optical microscopy, and X-ray microcomputed tomography to visualize and reconstruct the fossil cone in 3D. RESULTS: The analyses demonstrate unequivocally that the sporophylls of the new Mazon Creek cone are arranged in whorls of six and have characters typical of Sphenophyllales, including epidermal cells with undulatory margins and in situ spores assignable to Columinisporites. The combination of characters, including sporophyll arrangement, anatomy, and spore type, supports the establishment of Hexaphyllostrobus kostorhysii gen. et sp. nov. within Sphenophyllales. Furthermore, we show that Tetraphyllostrobus, although originally described as possessing smooth monolete spores, actually possesses Columinisporites-type spores, indicating that it, too, was most likely a sphenophyll. CONCLUSIONS: The recognition of Hexaphyllostrobus contributes to our knowledge of Pennsylvanian sphenophyll diversity, and in particular increases the number of species with in situ Columinisporites-type spores. Attribution of Hexaphyllostrobus to Sphenophyllales calls into question current interpretations of Tetraphyllostrobus suggesting that future research on better-preserved macrofossil material may demonstrate a sphenophyllalean relationship.

A permineralized Early Cretaceous lycopsid from China and the evolution of crown clubmosses.

Lycopodiaceae are one of three surviving families of lycopsids, a lineage of vascular plants with a fossil history dating to at least the Early Devonian or perhaps the Late Silurian (c. 415 Ma). Many fossils have been linked to crown Lycopodiaceae, but the lack of well-preserved material has hindered definitive recognition of this group in the paleobotanical record. New, exceptionally well-preserved permineralized lycopsid fossils from the Early Cretaceous (125.6 ± 1.0 Ma) of Inner Mongolia, China, were examined in detail using acetate peel and micro-computed tomography techniques. The anatomy of extant Lycopodiaceae was analyzed for comparison using fluorescence microscopy. Phylogenetic relationships of the new fossil to extant Lycopodiaceae were evaluated using parsimony and maximum likelihood analyses. Lycopodicaulis oellgaardii gen. et sp. nov. provides the earliest unequivocal and best-documented evidence of crown Lycopodiaceae and Lycopodioideae, based on anatomically-preserved fossil material. Recognition of Lycopodicaulis in Asia during the Early Cretaceous indicates the presence of crown Lycopodiaceae at this time, and striking similarities of stem anatomy with extant species provide a framework for the understanding of the interaction of branching and vascular anatomy in crown-group lycopsids.

Early Cretaceous abietoid Pinaceae from Mongolia and the history of seed scale shedding.

PREMISE: Seed cones of extant Pinaceae exhibit two mechanisms of seed release. In "flexers" the cone scales remain attached to the central axis, while flexing and separating from each other to release the seeds. In "shedders" scales are shed from the axis, with the seeds either remaining attached to the scale or becoming detached. The early fossil history of Pinaceae from the Jurassic to Early Cretaceous is dominated by flexing seed cones, while the systematic information provided by shedding fossil cones has been overlooked and rarely integrated with data based on compression and permineralized specimens. We describe the earliest and best-documented evidence of a "shedder" seed cone from the Aptian-Albian of Mongolia. METHODS: Lignite samples from Tevshiin Govi locality were disaggregated in water, washed, and dried in air. Fossils were compared to material of extant Pinaceae using LM and CT scans. RESULTS: Lepidocasus mellonae gen. et sp. nov. is characterized by a seed cone that disarticulated at maturity and shed obovate bract-scale complexes that have a distinctive ribbed surface and an abaxial surface covered with abundant trichomes. The ovuliferous scale has ca. 30-40 resin canals, but only scarce xylem near the attachment to the cone axis. Resin vesicles are present in the seed integument. Phylogenetic analysis places Lepidocasus as sister to extant Cedrus within the abietoid grade. CONCLUSIONS: The exquisite preservation of the trichomes in L. mellonae raises questions about their potential ecological function in the cones of fossil and living Pinaceae. Lepidocasus mellonae also shows that a shedding dispersal syndrome, a feature that has often been overlooked, evolved early in the history of Pinaceae during the Early Cretaceous.

Exceptional preservation of tiny embryos documents seed dormancy in early angiosperms.

The rapid diversification of angiosperms through the Early Cretaceous period, between about 130-100 million years ago, initiated fundamental changes in the composition of terrestrial vegetation and is increasingly well understood on the basis of a wealth of palaeobotanical discoveries over the past four decades and their integration with improved knowledge of living angiosperms. Prevailing hypotheses, based on evidence both from living and from fossil plants, emphasize that the earliest angiosperms were plants of small stature with rapid life cycles that exploited disturbed habitats in open, or perhaps understorey, conditions. However, direct palaeontogical data relevant to understanding the seed biology and germination ecology of Early Cretaceous angiosperms are sparse. Here we report the discovery of embryos and their associated nutrient storage tissues in exceptionally well-preserved angiosperm seeds from the Early Cretaceous. Synchrotron radiation X-ray tomographic microscopy of the fossil embryos from many taxa reveals that all were tiny at the time of dispersal. These results support hypotheses based on extant plants that tiny embryos and seed dormancy are basic for angiosperms as a whole. The minute size of the fossil embryos, and the modest nutrient storage tissues dictated by the overall small seed size, is also consistent with the interpretation that many early angiosperms were opportunistic, early successional colonizers of disturbance-prone habitats.

The presumed ginkgophyte Umaltolepis has seed-bearing structures resembling those of Peltaspermales and Umkomasiales.

The origins of the five groups of living seed plants, including the single relictual species Ginkgo biloba, are poorly understood, in large part because of very imperfect knowledge of extinct seed plant diversity. Here we describe well-preserved material from the Early Cretaceous of Mongolia of the previously enigmatic Mesozoic seed plant reproductive structure Umaltolepis, which has been presumed to be a ginkgophyte. Abundant new material shows that Umaltolepis is a seed-bearing cupule that was borne on a stalk at the tip of a short shoot. Each cupule is umbrella-like with a central column that bears a thick, resinous, four-lobed outer covering, which opens from below. Four, pendulous, winged seeds are attached to the upper part of the column and are enclosed by the cupule. Evidence from morphology, anatomy, and field association suggests that the short shoots bore simple, elongate Pseudotorellia leaves that have similar venation and resin ducts to leaves of living GinkgoUmaltolepis seed-bearing structures are very different from those of Ginkgo but very similar to fossils described previously as Vladimaria. Umaltolepis and Vladimaria do not closely resemble the seed-bearing structures of any living or extinct plant, but are comparable in some respects to those of certain Peltaspermales and Umkomasiales (corystosperms). Vegetative similarities of the Umaltolepis plant to Ginkgo, and reproductive similarities to extinct peltasperms and corystosperms, support previous ideas that Ginkgo may be the last survivor of a once highly diverse group of extinct plants, several of which exhibited various degrees of ovule enclosure.

Tanispermum, a new genus of hemi-orthotropous to hemi-anatropous angiosperm seeds from the Early Cretaceous of eastern North America.

PREMISE OF THE STUDY: Exotestal seeds with characters that indicate relationship to extant Austrobaileyales and Nymphaeales are abundant in Early Cretaceous sediments from Portugal and eastern North America, but their variety and unique features provide evidence of extensive extinct diversity among early angiosperms. METHODS: The fossils were extracted from Early Cretaceous sediments from Virginia and Maryland, United States, by sieving in water. After cleaning with HF, HCl and water, they were examined using SEM and SRXTM and compared to seeds of extant and fossil angiosperms. KEY RESULTS: A new genus, Tanispermum gen. nov., with four species (T. hopewellense sp. nov., T. marylandense sp. nov., T. drewriense sp. nov., and T. antiquum sp. nov.) is recognized. Relationship with extant Austrobaileyales and Nymphaeales is indicated by an exotesta of tall palisade sclerenchyma with undulate anticlinal walls forming a jigsaw puzzle-like surface pattern. However, seeds of Tanispermum differ from those of Austrobaileyales and Nymphaeales in their hemi-orthotropous-hemi-anatropous organization. Attempts to place Tanispermum in a phylogenetic context confront a variety of problems, including lack of information on other parts of these extinct plants. CONCLUSIONS: The discovery highlights the extent to which the morphology of extant angiosperms is not representative of the diversity that once existed among early-diverging members of the group. The relictual nature of extant taxa near the base of the angiosperm tree greatly complicates the reconstruction of ancestral character states and emphasizes the need for focused paleobotanical studies to elucidate the extinct diversity that existed early in angiosperm evolution.

An exquisitely preserved filmy fern (Hymenophyllaceae) from the Early Cretaceous of Mongolia.

PREMISE OF THE STUDY: Hymenophyllaceae ("filmy ferns") are a widely distributed group of predominantly tropical, epiphytic ferns that also include some temperate and terrestrial species. Hymenophyllaceae are one of the earliest-diverging lineages within leptosporangiate ferns, but their fossil record is sparse, most likely because of their low fossilization potential and commonly poor preservation of their delicate, membranaceous fronds. A new species of unequivocal fossil Hymenophyllaceae, Hymenophyllum iwatsukii sp. nov., is described from the Early Cretaceous of Mongolia based on abundant and exceptionally well-preserved material. METHODS: Bulk lignite samples collected from Tevshiin Govi and Tugrug localities in Mongolia, were disaggregated in water, cleaned with hydrochloric and hydrofluoric acids, washed, and dried in air. Fossils were examined and compared to material of extant Hymenophyllaceae using LM and SEM. KEY RESULTS: The fossil fern specimens are assigned to the Hymenophyllaceae based on their membranaceous laminae with marginal sori that have sessile to short-stalked sporangia with oblique, complete annuli, and trilete, tetrahedral-globose spores. Within the family, the fossil material is assigned to the extant genus Hymenophyllum on the basis of bivalvate indusia and short, included receptacles. CONCLUSIONS: Hymenophyllum iwatsukii was likely an epiphyte based on the sedimentary environment in which the fossils are preserved, the associated fossil flora, and the growth habit of extant species of Hymenophyllum. The new fossil species underlines the extent to which morphological characters in Hymenophyllum have been conserved despite significant tectonic, climatic, ecological, and floristic changes since the Early Cretaceous.

A Consensus-Driven Agenda for Emergency Medicine Firearm Injury Prevention Research.

STUDY OBJECTIVE: To identify critical emergency medicine-focused firearm injury research questions and develop an evidence-based research agenda. METHODS: National content experts were recruited to a technical advisory group for the American College of Emergency Physicians Research Committee. Nominal group technique was used to identify research questions by consensus. The technical advisory group decided to focus on 5 widely accepted categorizations of firearm injury. Subgroups conducted literature reviews on each topic and developed preliminary lists of emergency medicine-relevant research questions. In-person meetings and conference calls were held to iteratively refine the extensive list of research questions, following nominal group technique guidelines. Feedback from external stakeholders was reviewed and integrated. RESULTS: Fifty-nine final emergency medicine-relevant research questions were identified, including questions that cut across all firearm injury topics and questions specific to self-directed violence (suicide and attempted suicide), intimate partner violence, peer (nonpartner) violence, mass violence, and unintentional ("accidental") injury. Some questions could be addressed through research conducted in emergency departments; others would require work in other settings. CONCLUSION: The technical advisory group identified key emergency medicine-relevant firearm injury research questions. Emergency medicine-specific data are limited for most of these questions. Funders and researchers should consider increasing their attention to firearm injury prevention and control, particularly to the questions identified here and in other recently developed research agendas.

The emergence of core eudicots: new floral evidence from the earliest Late Cretaceous.

Eudicots, the most diverse of the three major clades of living angiosperms, are first recognized in the latest Barremian-earliest Aptian. All Early Cretaceous forms appear to be related to species-poor lineages that diverged before the rise of core eudicots, which today comprise more than 70% of angiosperm species. Here, we report the discovery of a well-preserved flower, Caliciflora mauldinensis, from the earliest Late Cretaceous, with unequivocal core eudicot features, including five sepals, five petals and two whorls of stamens borne on the rim of a floral cup containing three free carpels. Pollen is tricolporate. Carpels mature into follicular fruitlets. This character combination suggests a phylogenetic position among rosids, but more specific assignment is precluded by complex patterns of character evolution among the very large number of potentially relevant extant taxa. The whorled floral organization is consistent with ideas that this stable pattern evolved early and was a prerequisite for more integrated patterns of floral architecture that evolved later. However, limited floral synorganization in Caliciflora and all earlier eudicot flowers recognized so far, calls into question hypotheses that substantial diversification of core eudicots had already occurred by the end of the Early Cretaceous.

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.

Heterogeneous Rates of Molecular Evolution and Diversification Could Explain the Triassic Age Estimate for Angiosperms.

Dating analyses based on molecular data imply that crown angiosperms existed in the Triassic, long before their undisputed appearance in the fossil record in the Early Cretaceous. Following a re-analysis of the age of angiosperms using updated sequences and fossil calibrations, we use a series of simulations to explore the possibility that the older age estimates are a consequence of (i) major shifts in the rate of sequence evolution near the base of the angiosperms and/or (ii) the representative taxon sampling strategy employed in such studies. We show that both of these factors do tend to yield substantially older age estimates. These analyses do not prove that younger age estimates based on the fossil record are correct, but they do suggest caution in accepting the older age estimates obtained using current relaxed-clock methods. Although we have focused here on the angiosperms, we suspect that these results will shed light on dating discrepancies in other major clades.

Hemisphere-scale differences in conifer evolutionary dynamics.

Fundamental differences in the distribution of oceans and landmasses in the Northern and Southern Hemispheres potentially impact patterns of biological diversity in the two areas. The evolutionary history of conifers provides an opportunity to explore these dynamics, because the majority of extant conifer species belong to lineages that have been broadly confined to the Northern or Southern Hemisphere during the Cenozoic. Incorporating genetic information with a critical review of fossil evidence, we developed an age-calibrated phylogeny sampling ∼80% of living conifer species. Most extant conifer species diverged recently during the Neogene within clades that generally were established during the later Mesozoic, but lineages that diversified mainly in the Southern Hemisphere show a significantly older distribution of divergence ages than their counterparts in the Northern Hemisphere. Our tree topology and divergence times also are best fit by diversification models in which Northern Hemisphere conifer lineages have higher rates of species turnover than Southern Hemisphere lineages. The abundance of recent divergences in northern clades may reflect complex patterns of migration and range shifts during climatic cycles over the later Neogene leading to elevated rates of speciation and extinction, whereas the scattered persistence of mild, wetter habitats in the Southern Hemisphere may have favored the survival of older lineages.

Cone size is related to branching architecture in conifers.

The relationship between branch diameter and leaf size has been widely used to understand how vegetative resources are allocated in plants. Branching architecture influences reproductive allocation as well, but fewer studies have explored this relationship at broad phylogenetic or ecological scales. In this study, we tested whether pollen-producing and seed-producing cone size scales with branch diameter in conifers, a diverse and globally distributed lineage of nonflowering seed plants. Branch diameter and cone size were analyzed using multiple regression models and evolutionary models of trait evolution for a data set of 293 extant conifer species within an explicit phylogenetic framework. Branch diameter is a strong predictor of cone size across conifer species, particularly for pollen cones and dry seed cones. However, these relationships are complex in detail because leaf morphology and seed dispersal biology influence the specific ways in which they are expressed. The ubiquity and strength of these scaling relationships across conifers suggest that reproductive and vegetative morphologies are coupled in the group, and it is therefore difficult to disentangle the evolution of cone size from the evolution of branching architecture.

The floral morphospace--a modern comparative approach to study angiosperm evolution.

Morphospaces are mathematical representations used for studying the evolution of morphological diversity and for the evaluation of evolved shapes among theoretically possible ones. Although widely used in zoology, they--with few exceptions--have been disregarded in plant science and in particular in the study of broad-scale patterns of floral structure and evolution. Here we provide basic information on the morphospace approach; we review earlier morphospace applications in plant science; and as a practical example, we construct and analyze a floral morphospace. Morphospaces are usually visualized with the help of ordination methods such as principal component analysis (PCA) or nonmetric multidimensional scaling (NMDS). The results of these analyses are then coupled with disparity indices that describe the spread of taxa in the space. We discuss these methods and apply modern statistical tools to the first and only angiosperm-wide floral morphospace published by Stebbins in 1951. Despite the incompleteness of Stebbins' original dataset, our analyses highlight major, angiosperm-wide trends in the diversity of flower morphology and thereby demonstrate the power of this previously neglected approach in plant science.