Permian ginkgophyte fossils from the Dolomites resemble extant O-ha-tsuki aberrant leaf-like fructifications of Ginkgo biloba L
© Fischer et al; licensee BioMed Central Ltd. 2010
Received: 11 May 2010
Accepted: 3 November 2010
Published: 3 November 2010
Structural elucidation and analysis of fructifications of plants is fundamental for understanding their evolution. In case of Ginkgo biloba, attention was drawn by Fujii in 1896 to aberrant fructifications of Ginkgo biloba whose seeds are attached to leaves, called O-ha-tsuki in Japan. This well-known phenomenon was now interpreted by Fujii as being homologous to ancestral sporophylls. The common fructification of Ginkgo biloba consists of 1-2 (rarely more) ovules on a dichotomously divided stalk, the ovules on top of short stalklets, with collars supporting the ovules. There is essentially no disagreement that either the whole stalk with its stalklets, collars and ovules is homologous to a sporophyll, or, alternatively, just one stalklet, collar and ovule each correspond to a sporophyll. For the transition of an ancestral sporophyll resembling extant O-ha-tsuki aberrant leaves into the common fructification with stalklet/collar/ovule, evolutionary reduction of the leaf lamina of such ancestral sporophylls has to be assumed. Furthermore, such ancestral sporophylls would be expected in the fossil record of ginkgophytes.
From the Upper Permian of the Bletterbach gorge (Dolomites, South Tyrol, Italy) ginkgophyte leaves of the genus Sphenobaiera were discovered. Among several specimens, one shows putatively attached seeds, while other specimens, depending on their state of preservation, show seeds in positions strongly suggesting such attachment. Morphology and results of a cuticular analysis are in agreement with an affiliation of the fossil to the ginkgophytes and the cuticle of the seed is comparable to that of Triassic and Jurassic ones and to those of extant Ginkgo biloba. The Sphenobaiera leaves with putatively attached seeds closely resemble seed-bearing O-ha-tsuki leaves of extant Ginkgo biloba. This leads to the hypothesis that, at least for some groups of ginkgophytes represented by extant Ginkgo biloba, such sporophylls represent the ancestral state of fructifications.
Some evidence is provided for the existence of ancestral laminar ginkgophyte sporophylls. Homology of the newly found fossil ginkgophyte fructifications with the aberrant O-ha-tsuki fructifications of Ginkgo biloba is proposed. This would support the interpretation of the apical part of the common Ginkgo biloba fructification (stalklet/collar/ovule) as a sporophyll with reduced leaf lamina.
Fossil ginkgophyte fructifications are rare in the fossil record . Most closely related to Ginkgo biloba are ovulate structures attached to brachyblasts (spur shoots) carrying also Ginkgo-like vegetative leaves (or associations of both in the fossiliferous sediments) from the Mesozoic and Cenozoic: Ginkgo yimaensis (Jurassic) , Ginkgo apodes (Late Jurassic to Early Cretaceous) , and Ginkgo adiantoides leaves/Ginkgo geissertii ovule (Cenozoic) [9, 10]. Nehvizdyella bipartita (Late Cretaceous)  has similar fructifications, but non-divided leaves. The fossil taxa affiliated to Ginkgo can be ordered in an evolutionary series with successive reduction of individual stalks, number of ovules and accompanying increase in the size of ovules and in the width of the leaf segments, reflecting also ontogenetic sequences in Ginkgo biloba . Palaeozoic fructifications of ginkgophytes are also known, but much more difficult to interpret. Trichopitys heteromorpha from the Lower Permian  is the oldest generally accepted ginkgophyte [, and other authors]. It consists of axes carrying dichotomous, non-laminar leaves with zones of dividing stalks with terminal ovules which are possibly recurved. Karkenia is a genus with multi-ovulate axes or multi-ovulate globular fructifications; the first species (associated with Ginkgoites tigrensis leaves) was described by Archangelsky from the Lower Cretaceous of Patagonia . Another well known member is the Liassic Karkenia hauptmannii, represented by brachyblasts with globular fructifications and Sphenobaiera leaves . Avatia bifurcata and Hamshawvia longipedunculata from the Triassic of South Africa  also possess globular multi-ovulate fructifications (on dichotomous stalks). Hamshawvia fructifications are found attached to brachyblasts with Sphenobaiera vegetative leaves.
Recently, Naugolnykh  has reviewed "foliar" seed-bearing organs of Paleozoic ginkgophytes. Some of the taxa he included in the ginkgophytes, e.g., Arberia, have been interpreted differently i.e. as glossopterid . Taxa that can be accepted as ginkgophyte seed-bearing organs clearly have a dichotomous structure like Trichopitys, Karkenia, Toretzia, and Grenana. These genera show stalks carrying ovules. If accepted as being homologous to leaves (sporophylls), these either would represent non-laminar leaves, or, alternatively, the laminar part would have been already reduced. The latter possibility led Naugolnykh to suppose "pre-karkeniaceous" ancestors of ginkgophytes.
Here we report on Permian laminar ginkgophyte leaves with putatively attached seeds.
Description of the fossils from the Upper Permian of the Dolomites and comparison with ginkgophyte characters
Cuticles were prepared from the leaf lamina region of the counterpart of the first specimen (figure 3).
Comparison to cuticles of other ginkgophytes
Cuticles of extant Ginkgo biloba (O-ha-tsuki form)
Further specimens of Sphenobaiera leaves associated with seeds from the Bletterbach
The systematic position of the leaves with putatively attached seeds from the Bletterbach could principally be supposed among the cycads, the pteridosperms, or the ginkgophytes. Cycadophytes, however, are characterized by pinnately (or rarely bipinnately) compound leaves, most unlike the Bletterbach fossils. Among pteridosperms the character of interest "leaf-borne seeds" is found, but leaflets are mostly not dichotomous. There are Y-shaped fronds which, in case of forms with entire margin (non-pinnate) like the Triassic Dicoidium dutoitii, show similarity in gross morphology (, figure 15.20) to once-dichotomous leaves like PAL-1368, but not to the multiple-dichotomous forms of Sphenobaiera from the Bletterbach (figure 12). Most importantly, these leaves possess central veins (the rachis). In contrast, in well preserved Sphenobaiera specimens from the Bletterbach dichotomous/parallel venation can be observed; and a prominent midvein should be observable in PAL-1368 but is absent. The Permian pteridosperm Dichophyllum flabellifera (, figure 15.57) possesses divided pinnules similar to Sphenobaiera leaves, but pinnules are tongue-shaped and have midveins. Based on the described morphology an affiliation of PAL-1368 to the pteridosperms can be excluded. The leaves with putatively attached seeds can be identified as belonging to the ginkgophyte genus Sphenobaiera.
The results of the cuticle studies on PAL-1368 also support the ginkgophyte origin of the fossil leaf and seed. The differences of leaf and seed cuticles are comparable to these differences in modern Ginkgo biloba or its O-ha-tsuki form. The cuticle structures of the fossil seed and those of extant O-ha-tsuki ovules show similar isodiametric to rectangular cell shapes and idioblasts in both cases, which may represent aborted stomata, trichome bases or glands. For Ginkgo biloba leaves Florin  mentions the rare occurrence of trichomes. With the SEM it is visible, that the cuticle is covered with wax. The indistinct parts of the LM pictures are caused by that. Two tubular structures (SEM) assumed to consist of wax were observed, this has also been described for Ginkgo biloba and other gymnosperms . In contrast to the impression from the LM picture, the stomata are also surrounded by papillae on the surface. The fossil seed cuticle differs from the extant one only by the distinctly thicker anticlinal walls and the thickened subsidiary cells.
Cuticle structures especially of Permian Sphenobaiera leaves have only been described by Florin  from the Lower Permian of France (Sphenobaiera raymondi) and by Schweitzer  from the Upper Permian of Germany (Sphenobaiera digitata). The cuticle of the Sphenobaiera raymondi specimen is not well preserved. It seems to be composed mainly of isodiametric cells instead of longitudinal ones, possesses straight cell walls, and stomatal complexes are specified as haplocheilic with narrowing of the stomatal pore. Sphenobaiera digitata cuticles are described as amphistomatic, with a cuticle showing longitudinally oriented cells, rows of stomata towards the center of the lamina and less regular cells and stomata distribution towards the margin. Papillae are present only on subsidiary cells. Stomatal complexes are described as dicyclic, but seem to be mainly monocyclic as is shown in the drawing given in .
Fossil ginkgophyte leaves are often amphistomatic with rare stomata in the adaxial cuticle, but hypostomatic leaves have also been described  and have been found also among common Ginkgo biloba leaves as well as varying stomata abundances in apical and basal leaf parts. Furthermore, stomata index for Ginkgo biloba was reported to be inversely correlated with CO2 concentration . Varying stomata abundance is also described for a fossil ginkgophyte . Hypostomatic ginkgophyte leaves are represented by a Sphenobaiera species from the Triassic Molteno Formation in South Africa [, p. 133] and by Baiera cf. furcata from the Middle Jurassic of China . From the Jurassic Yorkshire Flora  Sphenobaiera ophioglossum shows similar cuticle structures (cell types of costal and intercostal fields, stomatal complexes, presence of papillae) [, figure 17 D], but is designated as the adaxial cuticle. The abaxial cuticle of Sphenobaiera schenckii from the Triassic of South Africa  is seemingly identical, but its adaxial cuticle shows abundant stomatal complexes (not present in the adaxial cuticle of the Bletterbach specimen). Several Sphenobaiera leaves from the Bletterbach have to be studied in future for detailed taxonomic comparisons and specific identification of these leaves.
With respect to the cuticle of the fossil seed depicted here, the cells of the outer cuticle of the integument of Ginkgo yimaensis ovules [7, plate 3, figure four] are very similar and the description also concurs. One distinct difference is the size of the stomatal complex, which is much larger (150-175 × 7.5-17.5 (-35) μm) and less circular than in the specimen from Bletterbach. The cuticle of the outer integument of the ovules, described for Yimaia recurva (associated with leaves of Baiera hallei)  differs by the rounded corners of the cells, the larger stomatal complexes and the unspecialized subsidiary cells. The cuticle of the Jurassic ovule Yimaia qinghaiensis  closely resembles that of the seed from the Bletterbach with respect to its general reticulate structure formed by irregular-polygonal and irregularly arranged cells, the absence of papillae on these cells, and its scattered stomatal complexes; even if any of these characters is not uncommon among gymnosperms. The Cretaceous ovuliferous organ Nehvizdyella bipartita  possesses the same cuticular characteristics as the Jurassic ovule Yimaia qinghaiensis and as the cuticle of the Sphenobaiera seed from Bletterbach. Ginkgo ginkgoidea (Tralau) Yang, Friis et Zhou  from the Jurassic of Sweden shows also alike cuticle structures.
Ontogenetic developmental aberrations can resemble primitive phylogenetic states of organs, commonly called "atavisms". Ideally, the aberrant character closely resembles the primitive character, which can either be known by the fossil record of the group, or by identification of the primitive state of the character by comparison with living representatives of sister groups and outgroups for the clade. For the aberrant O-ha-tsuki leaves of Ginkgo biloba the underlying genetics is completely unknown. An alternative interpretation of the phenomenon to atavism would be its description as a case of ectopic organ development, which offers no mechanistic explanation. Rothwell expected fossil occurrence of ginkgophyte brachyblasts (spur shoots) with sporophylls like the O-ha-tsuki leaves [, p.101]. Anderson and Anderson  also led attention to such "anomalous strobili with leafy expansions" (= O-ha-tsuki form) of Ginkgo biloba comparing them with their Triassic Avatia bifurcata fructification, even without a distinct laminar structure of the considered fossil. However, it can not be excluded that these Triassic fructifications represent rare aberrant forms.
Sphenobaiera represents a heterogeneous group of leaf fossils with wide stratigraphical distribution. The leaf morphogenus comprises dichotomous and wedge-shaped leaves, which do not possess a petiole like extant Ginkgo and the fossil leaf morphogenera Ginkgo, Baiera and Ginkgoites. Most species of Sphenobaiera are thought to represent ginkgophytes .
Zhou and Zheng  suggested an evolutionary scheme starting with Jurassic Ginkgo yimaensis with multiple small ovules and multiple divided vegetative leaves, subsequent reduction of ovule number and number of leaf segments in Cretaceous Ginkgo apodes and Cainozoic Ginkgo adiantoides, ending with Ginkgo biloba with predominantly only one ovule per stalk and bilobate to undivided vegetative leaves ("reduction hypothesis", "peramorphosis"). Given that the interpretation of the presented Permian fossils from Bletterbach as sporophylls is correct; these would represent an ancestral state of female ginkgophyte sporophylls before reduction of the sporophyll lamina had taken place, predating the reconstructed evolutionary series.
The Sphenobaiera fossils with seemingly attached seeds from the Upper Permian of the Bletterbach are hypothetically interpreted as ancestral ginkgophyte sporophylls with laminar structure, as would be expected from aberrant O-ha-tsuki leaves with seeds of extant Ginkgo biloba. A formal description or affiliation of the fossils at the species level will be provided together with the one for the numerous vegetative Sphenobaiera leaves and with the other floral elements. Ongoing excavations at the Bletterbach locality, at other Permian localities, but also screening of museum collections could provide more and possibly better preserved ginkgophyte leaves with ovules/seeds. Especially specimens with completely preserved vascular systems would be highly desirable for comparison with vegetative and fertile organs of extant Ginkgo biloba.
Excavation of the fossils and deposition
The fossils were recovered during excavations in the years 2003-2009. The figured specimens are kept in the palaeontological collection of the Museum of Nature South Tyrol (Bozen/Bolzano, figure three: no. PAL-1368, figure twelve: no. PAL-1369).
Small pieces of organic material were removed from the fossil with a scalpel, rinsed in water and treated with a drop of 10% HF until all sediment particles were dissolved. The material was rinsed in water and incubated in conc. HNO3 with KClO3 (Schulze's Reagent) until the organic material became transparent. The material was rinsed with water, briefly treated with 5% KOH, rinsed with water, and transferred into glycerol for study and storage. The samples of the O-ha-tsuki leaf and seed were taken from one specimen, from the middle part of the lower and upper half of the leaf and seed and directly put into Schulze's Reagent. One piece was studied with the LM, the other with the SEM. The O-ha-tsuki leaf sample for the LM has been stained with safranin. The cuticles were studied with a Nikon eclipse 80i light microscope (LM), the pictures had been taken with a Samsung digimax V70. For the scanning electron microscope FEI (SEM), the macerated wet cuticles were transferred to the SEM-stub with a carbon adhesive tape, where they dried. One piece of the fossil leaf cuticle (slide one) was removed from the glycerol, washed with water and ethanol and then picked onto carbon tape on the SEM-stub. During the drying process a rather distinct shrinkage of the cuticles has been observed, which results in partly smaller cell sizes of the SEM cuticles. Both, fossil and extant leaf cuticles are very thin and fragile; the cuticles of the seeds are thick. These thick cuticles tended to roll in and a strong mechanical pressure was necessary to get a nearly flat cuticle. The pictures were slightly adjusted with Adobe Photoshop 7.0 in brightness, contrast and frame.
Drawing of fossils
Drawings of fossils were performed using a binocular to discriminate biological structures from those produced by local destruction.
The specimens have been conserved in aqueous formaldehyde solution and are kept in the Herbarium of the Museum of Nature South Tyrol BOZ (PVASC15174).
The country South Tyrol is acknowledged for financial support for the excavations, the staff of the GeoCenter Aldein (Maria Pichler, Herta Obertegger, Christian Weber) for organizational support, and family Kalser (Aldein, South Tyrol) for their kind hospitality during our visits. Hiroshi Okada (Botanical Gardens, Osaka City University), the temple Mito Hachimanguu and Yoshiko Mashiko (Ibaraki-ken, Japan), Wolfgang Schwarz (Technische Universität München) and Josef Bogner (Ludwig-Maximilians-Universität München) are acknowledged for providing samples of O-ha-tsuki plant material or for their help to do so. We are grateful to Michael Krings (Ludwig-Maximilians-Universität München), Han van Konijnenburg-Van Cittert (Utrecht University) and Hans Kerp (Westfälische Wilhelms-Universität Münster) for critical discussion. Georg Janssen (Bayerische Staatssammlung für Paläontologie und Geologie) kindly provided photographies of the fossils. Stephan Schultka, Manfred Barthel (both Museum für Naturkunde, Leibniz-Institut für Evolutions- und Biodiversitätsforschung an der Humboldt-Universität zu Berlin) and Ralf Werneburg (Naturhistorisches Museum Schloss Bertholdsburg Schleusingen) are acknowledged for making fossils from the Lower Permian of Thuringia (not mentioned in the text) available to us for comparison. We acknowledge the proof-reading by Elizabeth Schroeder-Reiter (Ludwig-Maximilians-Universität München) as a native speaker and the efforts of two reviewers to improve our manuscript.
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