LETTERS Phase-contrast X-ray microtomography links Cretaceous seeds with Gnetales and Bennettitales Else Marie Friis 1 , Peter R. Crane 2 , Kaj Raunsgaard Pedersen 3 , Stefan Bengtson 1 , Philip C. J. Donoghue 4 , Guido W. Grimm 5 & Marco Stampanoni 6 Over the past 25 years the discovery and study of Cretaceous plant mesofossils has yielded diverse and exquisitely preserved fossil flowers that have revolutionized our knowledge of early angio- sperms 1 , but remains of other seed plants in the same mesofossil assemblages 2,3 have so far received little attention. These fossils, typically only a few millimetres long, have often been charred in natural fires and preserve both three-dimensional morphology and cellular detail. Here we use phase-contrast-enhanced synchrotron- radiation X-ray tomographic microscopy to clarify the structure of small charcoalified gymnosperm seeds from the Early Cretaceous of Portugal and North America. The new information links these seeds to Gnetales (including Erdtmanithecales, a putatively closely related fossil group 2 ), and to Bennettitales—important extinct Mesozoic seed plants with cycad-like leaves and flower-like repro- ductive structures. The results suggest that the distinctive seed architecture of Gnetales, Erdtmanithecales and Bennettitales defines a clade containing these taxa. This has significant conse- quences for hypotheses of seed plant phylogeny by providing sup- port for key elements of the controversial anthophyte hypothesis, which links angiosperms, Bennettitales and Gnetales. Relationships among extant seed plants (cycads, Ginkgo, conifers, Gnetales and angiosperms) remain controversial 4,5 , but because these are only a small sample of the total diversity in the seed plant clade, new information on extinct seed plants is likely to be crucial to resolve the current impasse 4–6 . Here we describe new material from a diverse collection of gymnospermous seeds that all have a similar basic structure. These seeds have excellent cellular preservation and are widespread in the Early Cretaceous mesofossil assemblages that we have examined from Portugal and eastern North America. They have been studied previously with standard techniques, but synchro- tron radiation X-ray tomographic microscopy, which has been applied successfully to study various microfossils without the need for destructive analysis 7–9 , provides a more complete understanding of their complex internal structure. For our material, attenuation- based synchrotron-radiation X-ray tomography (SRXTM) does not provide sufficient contrast because of the low absorption of the char- coalified cell walls, whereas phase-contrast X-ray tomographic microscopy (PCXTM, see Methods) provides high resolution at the cellular level. The fossil seeds are about 0.5–1.8-mm long, obovate to obtrian- gular or elliptical in longitudinal outline, and distinctly four-angled in transverse section (Figs 1, 2). At the apex there is a central projec- tion, which in some seeds is surrounded by four, pointed, tepal-like structures that project upwards from the four angles. The seeds have a broad stalk, but in most specimens it is broken off. The fossils super- ficially resemble angiosperm flowers 10 , but have also been compared with seeds of Gnetales and Erdtmanithecales 3,10 . PCXTM establishes that the fossils are small seeds comprised of two distinctly different layers surrounding the nucellus: an inner thin, membranous, integu- ment, formed by thin-walled cells, and a robust outer, sclerenchyma- tous, seed envelope that completely encloses the integument except for the micropylar opening. The integument is slightly shrunken in all specimens examined but there is no well-developed nucellus membrane or megaspore membrane. The integument and outer envelope are free for their full length and are attached only by a broad connection at the base (Fig. 1, left panel). The integument is free from the nucellus in its upper part and tapers into a long, narrow micropylar tube that is circular in transverse section. The micropylar canal is open towards the apex, and the integument in this region consists of one or two layers of small cells (Fig. 1, CS1). In the middle part and towards the base, the micropylar canal is closed by expanded tissue of the integument, which, at this level, consists of several cell layers. In particular, the cells of the inner epidermis are expanded radially to form a prom- inent ring that closes the micropylar canal (Fig. 1, CS2). At the base of the micropyle, the integument opens again, but the inner lining is irregular and the inner cells are broken down (Fig. 1, CS3). The envelope is mainly composed of a hard sclerenchymatous tissue. At the apex this tissue forms a distinct, central projection around the micropylar tube formed by the integument. It also forms the protrusions that extend from the corners of the seed (Fig. 1a–d). In some seeds these merge proximally to form a ring-like structure (Fig. 1a); in others they are thinner and more discrete. There is considerable diversity in seed size and the form of the outer surface among this group of seeds. The envelope has an inner layer of narrowly elongated scleren- chyma cells that are transversely aligned around the main body of the seed. Towards the apex these sclerenchyma cells are expanded radially (Figs 1d, 2c). The inner surface of the envelope is smooth and non-papillate, including around the micropyle (Figs 1d, 2c). The outer surface has irregular transverse ridges, which give the seed a wrinkled appearance (Figs 1b, c, 2a). The cells comprising this layer are elongated and aligned vertically. Occasional remains of paren- chymatous tissues outside the wrinkled surface of the sclerenchyma layer suggest that the seed envelope had a thin, fleshy outer covering. Only Gnetales (extant and extinct), Erdtmanithecales (extinct) and Bennettitales (extinct) are known to have seeds of this kind with an additional seed envelope and the integument extended into a long, narrow micropylar tube. In all three extant genera of Gnetales, the integument is thin and fused to the nucellus for most of its length. At the base, the fused integument is broadly attached and surrounded by one (Ephedra, Welwitschia) or two (Gnetum) envelopes, by which it is 1 Departments of Palaeobotany and Palaeozoology, Swedish Museum of Natural History, SE-104 05 Stockholm, Sweden. 2 Department of the Geophysical Sciences, University of Chicago, Chicago, Illinois 60637, USA. 3 Department of Earth Sciences, University of Aarhus, DK-8000 A ˚ rhus C, Denmark. 4 Department of Earth Sciences, University of Bristol, Bristol BS8 1RJ, UK. 5 Institute of Geosciences, Eberhard-Karls-University, D-72076 Tu ¨bingen, Germany. 6 Swiss Light Source, Paul Scherrer Institut, CH-5232 Villigen, Switzerland. Vol 450 | 22 November 2007 | doi:10.1038/nature06278 549 Nature ©2007 Publishing Group