First indisputable fossil Ilex (Aquifoliales: Aquifoliaceae) flower found in Baltic amber

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Rasmussen, Bo Johansen, Kathleen Dollman, Erik Wisaeus, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4958893/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract The genus Ilex (holly) in the monotypic family Aquifoliaceae contains more than 600 species distributed worldwide. Pollen fossils and macrofossils of fruits of Ilex are known from the late Cretaceous (90 Ma) and verified leaves from upper Miocene (10 Ma), but until now only a few more or less incomplete flower fossils have been suggested as referable to this genus. This paper reviews the fossil flowers earlier attributed to Ilex and presents a complete and indisputable staminate Ilex flower from Baltic amber (34–38 Ma) examined in fine detail with Synchrotron X-ray tomography. This find may challenge the hypothesis of a late Neogene dispersal of the genus from a center of origin in subtropical East Asia. Biological sciences/Evolution Biological sciences/Plant sciences Figures Figure 1 Figure 2 Introduction Ilex is the most species-rich genus of dioecious woody plants and is estimated to contain more than 600 extant species [ 1 ]. The genus is of considerable horticultural and economic interest. The leaves of more than 60 species of Ilex are used for beverages and several species are known for medicinal properties. The evergreen branches with red fruits of I. aquifolium and I. chinensis are widely applied symbols of Christmas and New Year in many parts of the world [ 2 ]. Ilex is well represented in the fossil record as pollen [ 3 ] as well as macrofossils, e.g., fruit stones [ 4 ] and leaves [5 ]. However, only six fossil species have been discussed as possible Ilex -flowers, all inclusions in Baltic amber published in the 19th century. Five of the six species are described and beautifully illustrated in colour by Conwentz in 1886 [ 6 ], all reproduced and enlarged in black and white in Schenck in 1890 [ 7 ]. Ilex prussica was mentioned informally by Caspary in 1872 [ 8 ] and validated as I. prussica Casp. ex Conw. by Conwentz [ 6 ] who added another fossil species, Ilex minuta Conw. [ 6 ]. The illustrations show two tetramerous flowers including pedicel (pubescent in both species) and calyx (marginally pubescent in I. minuta ). Ilex prussica is 5 mm high, I. minuta c.2.5 mm from the base of the flower to the apex of the stamens. The petals appear to be free but may be united slightly at the base. The petals are upright, with slightly incurved apices, in I. prussica ovate, in I. minuta spatulate. The stamens are significantly longer than the petals. The stamens appear to be opposite the petals, but accompanying floral diagrams indicates that they are alternating with the petals. A pistil or pistillodium cannot be seen. Conwentz’s contemporary colleague Loesener [ 9 ] did not accept these species as Ilex because of the small calyx and narrow floral base - they seem to lack a pistillodium which is present in all extant Ilex , and the stamen filaments are longer than the petals, a trait not seen in extant Ilex . Schenck [ 7 , 10 ] was also highly sceptical of the placement of the fossils in Ilex , but he did not suggest any other genera as possible extant relatives. Three detached corollas with epipetalous stamens were described in Ilex by Caspary [ 11 ]. Ilex aurita Casp. is hexamerous with unequal petal lobes. It was not depicted by Conwentz, who was in doubt about the determination. However, Loesener [ 9 ] accepted this fossil as an Ilex because of the overall similarity to extant species. Ilex minor Casp. is also hexamerous and was transferred to Sambucus as S. succinea by Conwents [ 6 ]. Loesener [ 9 ] disagreed and suggested that this could actually be the same species as I. aurita Casp. Ilex multiloba Casp. is another detached corolla, but with seven petal lobes. Conwentz transferred it to Sambucus multiloba (Casp.) Conw. [ 6 ] after having studied more material of what he considered the same species. Loesener suggested [ 9 ] it could be a teratological female Ilex flower because of the rather small anthers which might be staminodia. However, Schenck [ 7 ] accepted Conwentz’s placing of these fossils in Sambucus because of the shape of the corollas and the varying size and deviating number of petal lobes, a trait more common in Sambucus . A picture of a detached corolla embedded in amber and much resembling Sambucus succinea was shown on the front page and on p. 33 as “ Ilex sp.” by Bachofen-Echt [ 12 ] in 1949, but without scale or indication of collection or repository. Celastrinanthium hauchecornei (Celastraceae) is a tiny cyme with three tetramerous unopen buds, described and depicted by Conwentz [ 6 ]. Loesener noted [ 9 ] that it looked more like an Ilex , but decisive identification was not possible. It was accepted in Celastraceae by Schenck [ 7 ]. The original material of Ilex prussica and I. minutiflora was kept in the famous amber collection of the Westpreussische Provinzialmuseum, Danzig, which is supposed to have been lost during World War II [ 13 ]. The type of I. aurita belonged to the amber collector Kowalevski in Königsberg and is now in the collections of BGR (Bundesanstalt für Geologie und Rohstoffe, Berlin Spandau), but in poor condition [Sadowsky pers. comm. 2024 03 29]. The whereabouts of the original material of Sambucus succinea , S. multiloba and Celastrinanthium hauchecornei are uncertain. Material and methods The material studied is a piece of Baltic amber, roughly 80 x 70 x 15 mm (Supplementary Fig. S1 ), found at the West coast of Jutland, Denmark, near Hvide Sande, by the Danish amber enthusiast and collector Lars Wenneberg. Substantial amounts of amber is found along the shores of Denmark, assumed to have been redeposited during the quaternary ice age [ 14 ]. The piece contains several stellate oak hairs, a signature inclusion for Baltic amber, six arthropod fossils and a detached flower c. 3.2 mm in diameter. It is labelled NHMD 872634 at the Natural History Museum of Denmark and is nr. DK 1073 in the Danish “Danekræ” fossil trove [ 15 ], disallowing destructive methods of investigation. Imaging The inclusions were studied visually under a Leica MZ16A stereo microscope. Image stacks were taken with a Leica DFC420 digital camera and processed with Leica LAS X Life science microscope software and Zerene Stacker focus stacking software 1.04v. Synchrotron radiation propagation phase contrast micro-computed tomography of the entire amber piece was carried out at the European Synchrotron and Radiation Facility, Grenoble, France on beamline BM18. The Ilex flower and one syninclusion were scanned at 2µm. 3D image processing of the X-ray tomography dataset was conducted at the Danish Technological Institute using Bruker CTAn v. 1.18.8.0 software and the Trainable Weka Segmentation tool v. 3.3.2 in Fiji software v. 2.9.0. 3D visualisations were generated using Bruker CTVox software v. 3.3.0, enhanced with RGBA transfer functions to highlight different features of the flower. Sub-datasets of each stamen were added and separately coloured to enhance visualisations. See Supplementary methods S2 for synchrotron scanning and 3D image processing details. The drawing of the reconstructed flower (Fig. 2 D) was done from the light microscopy and CT images, CT-video clip (Supplementary video S3) and a 3D print from 3D dataset. Results Systematic palaeontology. Campanulids clade Order: Aquifoliales Senft Family: Aquifoliaceae Bercht. & J. Presl. Genus: Ilex L. Ilex wennebergii Rasmussen & B. Johans. sp. nov. Holotype: NHMD 872634 (DK 1073). Collected by Lars Wenneberg on the west coast of Jutland 2.5 km south of Hvide Sande, Denmark. Stratigraphic age . Estimates of the age of Baltic amber vary considerably, the most recent studies support a late Eocene (Priabonian) to the beginning of the Oligocene epoch 38 − 34 Ma [ 16 , 17 ] Etymology. The epithet honours the amber enthusiast and collector Lars Wenneberg, who has contributed to the knowledge of the biodiversity of Baltic amber with several interesting specimens. Diagnosis. Generally similar to the male flower of Ilex aquifolia L., but about half the size of that species. The ratio filament:anther is 1:1, while in I. aquifolia its is 3:1. Differs from I. prussica Caspary ex Conwentz and Ilex minuta Conwentz by the larger calyx and by the glabrous calyx, petals and filaments, wider flower base and stamens being shorter than the petals. Differs from I. aurita Caspary by being tetramerous. Description. A single staminate flower, embedded in amber at a late stage of flowering, all stamens open with filaments bending inwards (Fig. 1 a, b, d, f) and corolla partially detached from the receptacle (Fig. 1 c, e). The flower is c. 3.2 mm in diameter, with a calyx of four basically connate sepals with broadly triangular free lobes 0.6 × 0.6 mm (Fig. 2 a). Petals four, valvate, strongly concave and obovate, 1.3-2.0 mm long and 1.5 mm wide, basically connate forming a corolla and adjoined with the calyx tube. Stamens four, alternating with the petals, 1.5 mm long, filaments strap shaped, inserted into the corolla, about as long as the anthers. Anthers dorsifixed, with four thecae, opening by longitudinal slits. Rudimentary ovary (pistillodium) subglobose, apex obtuse, 0.4 mm high and 0.5 mm in diameter (Fig. 2 b, c). Remarks. The specimen is mostly an impression, the sepals, three petal, the stamens and even the carpels of the pistillodium are voids with scattered grains of degraded organic material. This is the usual state of amber inclusions [ 14 ]. Only the pistillodium and one petal appear to be more solid, filled out with a grainy substance (Fig. 2 b-c). The flower is withered, the corolla is partly detached, and the anthers are collapsed (Fig. 1 c, d, e, Supplementary Video S3). The wide open and empty anthers suggest that this is due to deflowering and not a result of the pedicel being broken or torn. A single supposed pollen grain with three colpi was observed floating near an anther but the CT scanning cannot resolve the surface texture and the flower is too deeply embedded to use high magnification light microscopy. There are no traces of hairs on the calyx lobes or petals, but stellate oak hairs of various size are attached to the surface of some of the petals and scattered in the amber close to the flower. A few air bubbles are attached to the sepals and petals and a larger one (c. 0.5 mm broad) is attached to the side and top of the pistillodium. Figure 2 d is a reconstruction of the flower before deflowering and embedding. Syninclusions. The amber piece NHMD 872634 (Danekræ DK 1073) contains six arthropod fossils: a stalk-eyed fly (Diptera: Diopsidae), another fly (Diptera), a braconid wasp (Hymenoptera: Braconidae), a click beetle (Coleoptera: Elateridae), a bristly millipede (Diplopoda: Polyxenida) and a mite (Acari). Discussion The present authors agree with Loesener [ 9 ] that I. prussica and I. minuta most likely do not belong to Ilex because of the apparent lack of pistillodium and stamens that are significantly longer than the petals. Tetramerous staminate flowers are found in a number of extant plant families; it is not possible to suggest a plausible alternative identification from the illustrations alone. The illustration of Celastrinanthium hauchecornei [ 6 ] does indeed look like a fragment of an Ilex inflorescence. If the material could be found a CT scanning of the unopened buds might reveal the family identity. Ilex aurita, I. minor (= Sambucus succinea) , and I . multiloba (= Sambucus multiloba ) are just floral fragments. Without calyx and pistillodium any identification is conjectural. However, the type of I. aurita is located in BGR, Berlin. If the anthers are preserved and pollen can be retrieved the family may be identified. The comments by Loesener [ 9 ] and Schenck [ 7 , 10 ] seem to be the last time these fossils were critically assessed in the literature, despite the considerable interest in the phylogeny and paleogeography of Ilex . Loesener later monographed the genus [ 18 , 19 ], but did not comment further on the alleged Ilex fossils. The names of the species are listed in some textbooks and catalogues of plant fossils [ 20 , 21 , 22 , 23 ], b ut Conwentz [ 6 ] is just mentioned en passant in one modern paper on the history of Ilex [ 24 ]. The cosmopolitan but very unequal extant distribution and the good fossil record, although without verifiable floral specimens, has made the genus a favourite object for the study of how clades speciate, spread and establish distributional areas [ 1, 3, 24, 25, 26, 27]. However, recent estimates of the evolutionary chronology and views of Ilex paleogeography rely mostly on the distinct pollen fossils [ 5 , 28 ]; and molecular clock calibrations of Ilex on a fruit stone from the Cretaceous and Miocene leaf fossils [ 1 , 26 ]. The latest estimate of the age the most recent common ancestor of extant Ilex is early Eocene (50.8 Ma) with an origin in moist subtropical East Asia and much later migrations to other areas of extant distributions – migration to Western Asia and Europe is calculated to have taken place in late Neogene about 5.8 Ma (4.35–7.61) [ 1 ]. Fossils older than early Eocene or found outside the areas of extant Ilex (e.g. Australia [ 29]), or from outside East Asia before the calculated time of migration to the finding place are thus assumed to represent lineages now extinct, perhaps as consequence of the late Miocene global cooling and drying [ 1 ]. The genus Ilex is remarkable for its highly uniform flower morphology [ 2 ], and it is possible that this has not varied much throughout its evolutionary history. It suggests that the floral biology of Ilex reached an early adaptive plateau and later contributed little to diversification [ 1 ]. The flower described here is very similar to extant staminate Ilex flowers, but I. wenne bergii and any other Ilex fossils from Baltic amber could even then represent extinct branches from the stem group, the “false starts” of Yao & al. [ 1 ]. On the other hand, the climatic conditions and composition of “the Baltic amber forest” and of the assumed area of origin of the crown clade of Ilex are supposed to have been very similar [ 1 , 16 ] and this humid, warm-temperate to subtropical forest has covered large parts of the northern hemisphere in the Eocene [ 30 , 31 ]. The possibility that species from Baltic amber may be members of the crown clade of Ilex should not be excluded. Conclusion The presence of the genus Ilex in the Baltic amber flora is verified, and the floral uniformity of the genus is shown to apply also to its evolutionary history. Synchrotron X-ray tomography of amber fossils is demonstrated to be a powerful tool for non-destructive investigation of critical details obscured by cracks and extranofacts (bubbles and debris). Declarations Competing interests: The authors declare that they have no competing interests. Author Contribution FNR mediated the contact between the collector of the material and the Natural History Museum and nominated it for inclusion in the Danekræ fossil trove, reviewed the literature and wrote the manuscript. FNR and BJ examined the material visually, did the light microscopy imaging and described the new species. KD operated Beamline18 at ESRF and collected the data used for 3D imaging. EW did the image processing and created the CT pictures and video clip. LV is curator of the amber fossil collection at the Natural History Museum and identified the syninclusions.All authors contributed to the interpretation of the observations and reviewed the manuscript. Acknowledgement We wish to thank Dr Ib Friis, Natural History Museum of Denmark and Dr Louis Ronse de Craene, Royal Botanic Gardens, Edinburgh for discussions of the possible affinities of Ilex prussica and Ilex minuta. Dr Eva-Maria Sadowski, Museum für Naturkunde, Berlin is thanked for searching for the original material of the species described by Caspary and Conwentz. We acknowledge the European Synchrotron Radiation Facility (ESRF) for provision of synchrotron radiation facilities, and we would like to thank Paul Tafforeau for assistance and support in using beamline BM18.We are grateful to Dr Hanne N. Rasmussen, Department of Geosciences and Natural Resource Management, University of Copenhagen, for her skilful reconstruction and drawing of Ilex wennebergii as it is supposed to have looked before fossilization.This study was supported by a grant from Danish Technological Institutes performance contract 2021-2024, entered with the Danish Agency for Higher Education and Science, under The Ministry of Higher Education and Science Denmark. Data Availability The fossil specimen is part of the amber collection at the Natural History Museum of Denmark and the Danekræ fossil trove. All observations made during this study are included in this paper. Correspondence should be addressed to FNR. References Yao, X., Song, Y., Yang, J-B., Tan, Y. & Corlett, R. Phylogeny and biogeography of the hollies ( Ilex L., Aquifoliaceae). Plant. Syst. Evol. 59 , 73–82 (2021). Loizeau, P-A., Savolainen, V., Andrews, S., Barriera, G. & Spichiger, R. Aquifoliaceae. In The Families and Genera of Vascular Plants XIV, Flowering Plants, Eudicots (ed Kubitzski, K.) 31–36 (Springer, (2016). Loizeau, P. A., Barriera, G., Manen, J. F. & Broennimann, O. Towards an understanding of the distribution of Ilex L. (Aquifoliaceae) on a World-wide scale. Biologiske Skrifter . 55 , 501–520 (2005). Knobloch, E. & Mai, D. H. Monographie der Früchte und Samen in der Kreide von Mitteleuropa. Ustredni Ustav Geol. 47 , 5–219 (1986). Li, X., Sun, B., Xiao, L., Wu, J. & Lin, Z. Leaf macrofossils of Ilex protocornuta sp. nov. (Aquifoliaceae) from the Late Miocene of East China: Implications for palaeoecology. Rev. Palaeobot Palynol . 161 , 87–103 (2010). Conwentz, H. & Engelmann Die Flora des Bernsteins, Zweiter Band. Die Angiospermen des Bernsteins. (1886). Schenk, A. Dicotyleae in Schimper & Schenck Palæophytologie, II Abteilung in Handbuch der Paläontologie (ed. Zittel, K. A.) 595–847 (R. Oldenburg, 1890). Caspary, R. Privatsitzung am 4. October. Schriften Königl. Phys.-Ökon. Ges. Königsberg , 13 (Abth. 2, Sitzungsberichte) 15–19 (1872). Loesener, T. Vorstudien zu einer Monographie der Aquifoliaceen (Mesch und Lichtenfeld, 1890). Schenck, A. Die fossilen Pflanzenreste in Handbuch der Botanik (ed. Schenck, A.,) Vierter Band, Sonderdruck, 1-284 (Eduard Trewendt, 1888). Caspary, R. Ueber neue fossile Pflanzen der blauen Erde, d. h. des Bernsteins, des Schwarzharzes und des Braunharzes. Schriften Phys. -Ökon Ges Königsberg Sitzungsberichte : 22 , 22–25 (1881). Bachofen-Echt, A. Der Bernstein und seine Einschlüsse (Springer, 1949). Anonymous Westpreußisches Provinzial-Museum Danzig. (2023). https://de.wikipedia.org/wiki/Westpreu%C3%9Fisches_Provinzial-Museum_Danzig . Weitschat, W. & Wichard, W. Atlas of plants and animals in Baltic amber (Friedrich Pfeil, 2002). Lindow, B. The Danekræ fossil trove. (2024). https://samlinger.snm.ku.dk/en/danekrae/ Sadowski, E. M., Seyfullah, L. J., Schmidt, A. R. & Kunzmann, L. Conifers of the ‘Baltic amber forest’ and their palaeoecological significance. Stapfia . 106 , 1–73 (2017). Sadowski, E. M., Schmidt, A. R. & Kunzmann, L. The hyperdiverse conifer flora of the Baltic amber forest. Palaeontograph B . 304 , 1–148 (2022). Loesener, T. Monographia Aquifoliacerum. Pars I. Nova Acta Acad. Caes Leop -Carol German Nat. Cur . 78 , 1–598 (1901). Loesener, T. Monographia Aquifoliacerum. Pars II. Nova Acta Acad. Caes. Leop.-Carol. German. Nat. Cur. 89, 1-313 (1908). (1908). Gothan, W. Lehrbuch der Paläobotanik (Borntraeger, 1921). Czečzott, H. : The flora of the Baltic amber and its age. Praze Muzeum Ziemi 4, 119–146 (1961) (1961). Spahr, U. Systematischer Katalog und Bibliographie der Bernstein - und Kopal-Flora. Stuttgarter Beitr. Naturk Ser. B . 195 , 1–99 (1993). IFPNI, The international fossil plant names index. (2024). http://www.ifpni.org/index.htm Manen, J. F., Boulter, M. C. & Naciri-Graven, Y. The complex history of the genus Ilex L. (Aquifoliaceae): evidence from the comparison of plastid and nuclear DNA sequences and from fossil data. Plant. Syst. Evol. 235 , 79–98 (2002). Cuénoud, P. et al. Molecular phylogeny and biogeography of the genus Ilex L. (Aquifoliaceae). Ann. Bot. 85 , 111–122 (2000). Manen, J. F., Barriera, G., Loizeau, P. A. & Naciri, Y. The history of extant Ilex species (Aquifoliaceae): evidence of hybridization within a Miocene radiation. Mol. Phylogenet Evol. 57 , 961–977 (2010). Yao, X. & al.Chloroplast genome structure in Ilex (Aquifoliaceae). Sci. Rep. 6 , 28559. https://doi.org/10.1038/srep28559 Kvaček, Z., Teodoridis, V. & Wang, Q. Ilex geissertii sp. n. (Aquifoliaceae), a fossil ancestor of Ilex sect. Ilex in the upper Miocene and Pliocene of Europe. Rev. Palaeobot Palynol . 157 , 192–210 (2009). Martin, H. A. The history of Ilex (Aquifoliaceae) with special reference to Australia: Evidence from pollen. Aust. J. Bot. 25 , 655–673 (1977). Sadowski, E-M. Towards a new picture of the ‘Baltic amber forest’ - flora, habitat types, and palaeoecology. Dissertation, Georg-August-Universität Göttingen (2017). Utescher, T. & Mosbrugger, V. Eocene vegetation patterns reconstructed from plant. diversity — A global perspective. Palaeogeography, Palaeoclimatology, Palaeoecology 247, 243–271 (2007). Additional Declarations No competing interests reported. Supplementary Files S1S2SupplementaryFig1andMethods.docx Supplementary Figure S1 Supplementary Methods S2 S3SupplementaryVideo.mp4 Supplementary Video S3 Cite Share Download PDF Status: Published Journal Publication published 12 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 09 Oct, 2024 Reviews received at journal 30 Sep, 2024 Reviews received at journal 25 Sep, 2024 Reviewers agreed at journal 23 Sep, 2024 Reviewers agreed at journal 21 Sep, 2024 Reviewers invited by journal 20 Sep, 2024 Editor assigned by journal 20 Sep, 2024 Editor invited by journal 05 Sep, 2024 Submission checks completed at journal 04 Sep, 2024 First submitted to journal 22 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4958893","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":360866281,"identity":"a8c2ab1c-8133-40af-8eff-2e7c2140bd72","order_by":0,"name":"Finn N. 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NHMD 872634. (a) Light microscopy view from below through c. 2 mm amber, (b) view from above through c. 10 mm amber with cracks and bubbles, (c-f) synchrotron CT scans, flower seen from below, different colour tones have been added to the four stamens and oak hairs attached to the flower are coloured pale orange, air bubbles blue, (c) flower seen from below, (d) view from above, (e) side view, note partial detachment of corolla, (f) rotated view from above.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4958893/v1/6d0d98c4b0eb6e662ca12e71.jpeg"},{"id":66075687,"identity":"604c0cfe-2e64-4a02-a7a4-dd2d7b373e14","added_by":"auto","created_at":"2024-10-07 12:57:30","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":940921,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIlex wennebergii\u003c/em\u003e sp. nov. NHMD 872634. (A-D) synchrotron CT scans, colouring as in Fig. 1, (a) view of flower fossil from below, (b) view from below, cross section of calyx and pistillodium. Calyx lobes and carpels are empty voids, but the interior of the pistillodium is massive; (b-c) longisections of flower, showing the massive pistillodium. Left petal is cut obliquely, thus appearing thicker than it is; (d) reconstruction of supposed look of flower before withering and fossilization. The colouring is imaginative.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4958893/v1/4a2afe8a893050a92d1546ef.jpeg"},{"id":69286142,"identity":"a725131d-ae67-4e12-bd7b-044bb3faa95b","added_by":"auto","created_at":"2024-11-18 19:30:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1811476,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4958893/v1/56773923-e366-4319-9927-43d576beed93.pdf"},{"id":66075686,"identity":"6ce45203-22e0-4a1e-89bd-651be436fb0e","added_by":"auto","created_at":"2024-10-07 12:57:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":629102,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Methods S2\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"S1S2SupplementaryFig1andMethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-4958893/v1/ae0110b2ec30317b4f98af06.docx"},{"id":66075689,"identity":"51508947-0a8e-433b-8ddf-8817d5c3019a","added_by":"auto","created_at":"2024-10-07 12:57:31","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":40973860,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Video S3\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"S3SupplementaryVideo.mp4","url":"https://assets-eu.researchsquare.com/files/rs-4958893/v1/8f692adb437cc6708f1c2c3d.mp4"}],"financialInterests":"No competing interests reported.","formattedTitle":"First indisputable fossil Ilex (Aquifoliales: Aquifoliaceae) flower found in Baltic amber","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eIlex\u003c/em\u003e is the most species-rich genus of dioecious woody plants and is estimated to contain more than 600 extant species [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The genus is of considerable horticultural and economic interest. The leaves of more than 60 species of \u003cem\u003eIlex\u003c/em\u003e are used for beverages and several species are known for medicinal properties. The evergreen branches with red fruits of \u003cem\u003eI. aquifolium\u003c/em\u003e and \u003cem\u003eI. chinensis\u003c/em\u003e are widely applied symbols of Christmas and New Year in many parts of the world [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eIlex\u003c/em\u003e is well represented in the fossil record as pollen [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] as well as macrofossils, e.g., fruit stones [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and leaves [5 ]. However, only six fossil species have been discussed as possible \u003cem\u003eIlex\u003c/em\u003e-flowers, all inclusions in Baltic amber published in the 19th century. Five of the six species are described and beautifully illustrated in colour by Conwentz in 1886 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], all reproduced and enlarged in black and white in Schenck in 1890 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eIlex prussica\u003c/em\u003e was mentioned informally by Caspary in 1872 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and validated as \u003cem\u003eI. prussica\u003c/em\u003e Casp. ex Conw. by Conwentz [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] who added another fossil species, \u003cem\u003eIlex minuta\u003c/em\u003e Conw. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The illustrations show two tetramerous flowers including pedicel (pubescent in both species) and calyx (marginally pubescent in \u003cem\u003eI. minuta\u003c/em\u003e). \u003cem\u003eIlex prussica\u003c/em\u003e is 5 mm high, \u003cem\u003eI. minuta\u003c/em\u003e c.2.5 mm from the base of the flower to the apex of the stamens. The petals appear to be free but may be united slightly at the base. The petals are upright, with slightly incurved apices, in \u003cem\u003eI. prussica\u003c/em\u003e ovate, in \u003cem\u003eI. minuta\u003c/em\u003e spatulate. The stamens are significantly longer than the petals. The stamens appear to be opposite the petals, but accompanying floral diagrams indicates that they are alternating with the petals. A pistil or pistillodium cannot be seen. Conwentz\u0026rsquo;s contemporary colleague Loesener [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] did not accept these species as \u003cem\u003eIlex\u003c/em\u003e because of the small calyx and narrow floral base - they seem to lack a pistillodium which is present in all extant \u003cem\u003eIlex\u003c/em\u003e, and the stamen filaments are longer than the petals, a trait not seen in extant \u003cem\u003eIlex\u003c/em\u003e. Schenck [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] was also highly sceptical of the placement of the fossils in \u003cem\u003eIlex\u003c/em\u003e, but he did not suggest any other genera as possible extant relatives.\u003c/p\u003e \u003cp\u003eThree detached corollas with epipetalous stamens were described in \u003cem\u003eIlex\u003c/em\u003e by Caspary [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. \u003cem\u003eIlex aurita\u003c/em\u003e Casp. is hexamerous with unequal petal lobes. It was not depicted by Conwentz, who was in doubt about the determination. However, Loesener [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] accepted this fossil as an \u003cem\u003eIlex\u003c/em\u003e because of the overall similarity to extant species. \u003cem\u003eIlex minor\u003c/em\u003e Casp. is also hexamerous and was transferred to \u003cem\u003eSambucus\u003c/em\u003e as \u003cem\u003eS. succinea\u003c/em\u003e by Conwents [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Loesener [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] disagreed and suggested that this could actually be the same species as \u003cem\u003eI. aurita\u003c/em\u003e Casp. \u003cem\u003eIlex multiloba\u003c/em\u003e Casp. is another detached corolla, but with seven petal lobes. Conwentz transferred it to \u003cem\u003eSambucus multiloba\u003c/em\u003e (Casp.) Conw. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] after having studied more material of what he considered the same species. Loesener suggested [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] it could be a teratological female \u003cem\u003eIlex\u003c/em\u003e flower because of the rather small anthers which might be staminodia. However, Schenck [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] accepted Conwentz\u0026rsquo;s placing of these fossils in \u003cem\u003eSambucus\u003c/em\u003e because of the shape of the corollas and the varying size and deviating number of petal lobes, a trait more common in \u003cem\u003eSambucus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eA picture of a detached corolla embedded in amber and much resembling \u003cem\u003eSambucus succinea\u003c/em\u003e was shown on the front page and on p. 33 as \u0026ldquo;\u003cem\u003eIlex\u003c/em\u003e sp.\u0026rdquo; by Bachofen-Echt [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] in 1949, but without scale or indication of collection or repository.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCelastrinanthium hauchecornei\u003c/em\u003e (Celastraceae) is a tiny cyme with three tetramerous unopen buds, described and depicted by Conwentz [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Loesener noted [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] that it looked more like an \u003cem\u003eIlex\u003c/em\u003e, but decisive identification was not possible. It was accepted in Celastraceae by Schenck [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe original material of \u003cem\u003eIlex prussica\u003c/em\u003e and \u003cem\u003eI. minutiflora\u003c/em\u003e was kept in the famous amber collection of the Westpreussische Provinzialmuseum, Danzig, which is supposed to have been lost during World War II [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The type of \u003cem\u003eI. aurita\u003c/em\u003e belonged to the amber collector Kowalevski in K\u0026ouml;nigsberg and is now in the collections of BGR (Bundesanstalt f\u0026uuml;r Geologie und Rohstoffe, Berlin Spandau), but in poor condition [Sadowsky pers. comm. 2024 03 29]. The whereabouts of the original material of \u003cem\u003eSambucus succinea\u003c/em\u003e, \u003cem\u003eS. multiloba\u003c/em\u003e and \u003cem\u003eCelastrinanthium hauchecornei\u003c/em\u003e are uncertain.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e \u003cb\u003eThe material studied\u003c/b\u003e is a piece of Baltic amber, roughly 80 x 70 x 15 mm (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), found at the West coast of Jutland, Denmark, near Hvide Sande, by the Danish amber enthusiast and collector Lars Wenneberg. Substantial amounts of amber is found along the shores of Denmark, assumed to have been redeposited during the quaternary ice age [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The piece contains several stellate oak hairs, a signature inclusion for Baltic amber, six arthropod fossils and a detached flower c. 3.2 mm in diameter. It is labelled NHMD 872634 at the Natural History Museum of Denmark and is nr. DK 1073 in the Danish \u0026ldquo;Danekr\u0026aelig;\u0026rdquo; fossil trove [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], disallowing destructive methods of investigation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImaging\u003c/b\u003e The inclusions were studied visually under a Leica MZ16A stereo microscope. Image stacks were taken with a Leica DFC420 digital camera and processed with Leica LAS X Life science microscope software and Zerene Stacker focus stacking software 1.04v.\u003c/p\u003e \u003cp\u003eSynchrotron radiation propagation phase contrast micro-computed tomography of the entire amber piece was carried out at the European Synchrotron and Radiation Facility, Grenoble, France on beamline BM18. The \u003cem\u003eIlex\u003c/em\u003e flower and one syninclusion were scanned at 2\u0026micro;m. 3D image processing of the X-ray tomography dataset was conducted at the Danish Technological Institute using Bruker CTAn v. 1.18.8.0 software and the Trainable Weka Segmentation tool v. 3.3.2 in Fiji software v. 2.9.0. 3D visualisations were generated using Bruker CTVox software v. 3.3.0, enhanced with RGBA transfer functions to highlight different features of the flower. Sub-datasets of each stamen were added and separately coloured to enhance visualisations. See Supplementary methods S2 for synchrotron scanning and 3D image processing details.\u003c/p\u003e \u003cp\u003eThe drawing of the reconstructed flower (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) was done from the light microscopy and CT images, CT-video clip (Supplementary video S3) and a 3D print from 3D dataset.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSystematic palaeontology.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCampanulids clade\u003c/p\u003e \u003cp\u003eOrder: Aquifoliales Senft\u003c/p\u003e \u003cp\u003eFamily: Aquifoliaceae Bercht. \u0026amp; J. Presl.\u003c/p\u003e \u003cp\u003eGenus: \u003cem\u003eIlex\u003c/em\u003e L.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIlex wennebergii\u003c/em\u003e Rasmussen \u0026amp; B. Johans. sp. nov.\u003c/p\u003e \u003cp\u003eHolotype: NHMD 872634 (DK 1073). Collected by Lars Wenneberg on the west coast of Jutland 2.5 km south of Hvide Sande, Denmark.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStratigraphic age\u003c/b\u003e. Estimates of the age of Baltic amber vary considerably, the most recent studies support a late Eocene (Priabonian) to the beginning of the Oligocene epoch 38\u0026thinsp;\u0026minus;\u0026thinsp;34 Ma [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003cb\u003eEtymology.\u003c/b\u003e The epithet honours the amber enthusiast and collector Lars Wenneberg, who has contributed to the knowledge of the biodiversity of Baltic amber with several interesting specimens.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDiagnosis.\u003c/b\u003e Generally similar to the male flower of \u003cem\u003eIlex aquifolia\u003c/em\u003e L., but about half the size of that species. The ratio filament:anther is 1:1, while in \u003cem\u003eI. aquifolia\u003c/em\u003e its is 3:1. Differs from \u003cem\u003eI. prussica\u003c/em\u003e Caspary ex Conwentz and \u003cem\u003eIlex minuta\u003c/em\u003e Conwentz by the larger calyx and by the glabrous calyx, petals and filaments, wider flower base and stamens being shorter than the petals. Differs from \u003cem\u003eI. aurita\u003c/em\u003e Caspary by being tetramerous.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDescription.\u003c/b\u003e A single staminate flower, embedded in amber at a late stage of flowering, all stamens open with filaments bending inwards (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b, d, f) and corolla partially detached from the receptacle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, e). The flower is c. 3.2 mm in diameter, with a calyx of four basically connate sepals with broadly triangular free lobes 0.6 \u0026times; 0.6 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Petals four, valvate, strongly concave and obovate, 1.3-2.0 mm long and 1.5 mm wide, basically connate forming a corolla and adjoined with the calyx tube. Stamens four, alternating with the petals, 1.5 mm long, filaments strap shaped, inserted into the corolla, about as long as the anthers. Anthers dorsifixed, with four thecae, opening by longitudinal slits. Rudimentary ovary (pistillodium) subglobose, apex obtuse, 0.4 mm high and 0.5 mm in diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRemarks.\u003c/b\u003e The specimen is mostly an impression, the sepals, three petal, the stamens and even the carpels of the pistillodium are voids with scattered grains of degraded organic material. This is the usual state of amber inclusions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Only the pistillodium and one petal appear to be more solid, filled out with a grainy substance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-c). The flower is withered, the corolla is partly detached, and the anthers are collapsed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d, e, Supplementary Video S3). The wide open and empty anthers suggest that this is due to deflowering and not a result of the pedicel being broken or torn. A single supposed pollen grain with three colpi was observed floating near an anther but the CT scanning cannot resolve the surface texture and the flower is too deeply embedded to use high magnification light microscopy. There are no traces of hairs on the calyx lobes or petals, but stellate oak hairs of various size are attached to the surface of some of the petals and scattered in the amber close to the flower. A few air bubbles are attached to the sepals and petals and a larger one (c. 0.5 mm broad) is attached to the side and top of the pistillodium. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed is a reconstruction of the flower before deflowering and embedding.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSyninclusions.\u003c/b\u003e The amber piece NHMD 872634 (Danekr\u0026aelig; DK 1073) contains six arthropod fossils: a stalk-eyed fly (Diptera: Diopsidae), another fly (Diptera), a braconid wasp (Hymenoptera: Braconidae), a click beetle (Coleoptera: Elateridae), a bristly millipede (Diplopoda: Polyxenida) and a mite (Acari).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present authors agree with Loesener [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] that \u003cem\u003eI. prussica\u003c/em\u003e and \u003cem\u003eI. minuta\u003c/em\u003e most likely do not belong to \u003cem\u003eIlex\u003c/em\u003e because of the apparent lack of pistillodium and stamens that are significantly longer than the petals. Tetramerous staminate flowers are found in a number of extant plant families; it is not possible to suggest a plausible alternative identification from the illustrations alone.\u003c/p\u003e \u003cp\u003eThe illustration of \u003cem\u003eCelastrinanthium hauchecornei\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] does indeed look like a fragment of an \u003cem\u003eIlex\u003c/em\u003e inflorescence. If the material could be found a CT scanning of the unopened buds might reveal the family identity.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIlex aurita, I. minor (=\u0026thinsp;Sambucus succinea)\u003c/em\u003e, and \u003cem\u003eI\u003c/em\u003e. \u003cem\u003emultiloba\u003c/em\u003e (=\u0026thinsp;\u003cem\u003eSambucus multiloba\u003c/em\u003e) are just floral fragments. Without calyx and pistillodium any identification is conjectural. However, the type of \u003cem\u003eI. aurita\u003c/em\u003e is located in BGR, Berlin. If the anthers are preserved and pollen can be retrieved the family may be identified.\u003c/p\u003e \u003cp\u003eThe comments by Loesener [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and Schenck [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] seem to be the last time these fossils were critically assessed in the literature, despite the considerable interest in the phylogeny and paleogeography of \u003cem\u003eIlex\u003c/em\u003e. Loesener later monographed the genus [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], but did not comment further on the alleged \u003cem\u003eIlex\u003c/em\u003e fossils. The names of the species are listed in some textbooks and catalogues of plant fossils [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], b ut Conwentz [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] is just mentioned en passant in one modern paper on the history of \u003cem\u003eIlex\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe cosmopolitan but very unequal extant distribution and the good fossil record, although without verifiable floral specimens, has made the genus a favourite object for the study of how clades speciate, spread and establish distributional areas [ 1, 3, 24, 25, 26, 27]. However, recent estimates of the evolutionary chronology and views of \u003cem\u003eIlex\u003c/em\u003e paleogeography rely mostly on the distinct pollen fossils [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]; and molecular clock calibrations of \u003cem\u003eIlex\u003c/em\u003e on a fruit stone from the Cretaceous and Miocene leaf fossils [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The latest estimate of the age the most recent common ancestor of extant \u003cem\u003eIlex\u003c/em\u003e is early Eocene (50.8 Ma) with an origin in moist subtropical East Asia and much later migrations to other areas of extant distributions \u0026ndash; migration to Western Asia and Europe is calculated to have taken place in late Neogene about 5.8 Ma (4.35\u0026ndash;7.61) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Fossils older than early Eocene or found outside the areas of extant \u003cem\u003eIlex\u003c/em\u003e (e.g. Australia [ 29]), or from outside East Asia before the calculated time of migration to the finding place are thus assumed to represent lineages now extinct, perhaps as consequence of the late Miocene global cooling and drying [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eIlex\u003c/em\u003e is remarkable for its highly uniform flower morphology [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and it is possible that this has not varied much throughout its evolutionary history. It suggests that the floral biology of \u003cem\u003eIlex\u003c/em\u003e reached an early adaptive plateau and later contributed little to diversification [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The flower described here is very similar to extant staminate Ilex flowers, but I. wenne\u003cem\u003ebergii\u003c/em\u003e and any other \u003cem\u003eIlex\u003c/em\u003e fossils from Baltic amber could even then represent extinct branches from the stem group, the \u0026ldquo;false starts\u0026rdquo; of Yao \u0026amp; al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. On the other hand, the climatic conditions and composition of \u0026ldquo;the Baltic amber forest\u0026rdquo; and of the assumed area of origin of the crown clade of \u003cem\u003eIlex\u003c/em\u003e are supposed to have been very similar [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and this humid, warm-temperate to subtropical forest has covered large parts of the northern hemisphere in the Eocene [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The possibility that species from Baltic amber may be members of the crown clade of \u003cem\u003eIlex\u003c/em\u003e should not be excluded.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe presence of the genus \u003cem\u003eIlex\u003c/em\u003e in the Baltic amber flora is verified, and the floral uniformity of the genus is shown to apply also to its evolutionary history. Synchrotron X-ray tomography of amber fossils is demonstrated to be a powerful tool for non-destructive investigation of critical details obscured by cracks and extranofacts (bubbles and debris).\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eFNR mediated the contact between the collector of the material and the Natural History Museum and nominated it for inclusion in the Danekr\u0026aelig; fossil trove, reviewed the literature and wrote the manuscript. FNR and BJ examined the material visually, did the light microscopy imaging and described the new species. KD operated Beamline18 at ESRF and collected the data used for 3D imaging. EW did the image processing and created the CT pictures and video clip. LV is curator of the amber fossil collection at the Natural History Museum and identified the syninclusions.All authors contributed to the interpretation of the observations and reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe wish to thank Dr Ib Friis, Natural History Museum of Denmark and Dr Louis Ronse de Craene, Royal Botanic Gardens, Edinburgh for discussions of the possible affinities of Ilex prussica and Ilex minuta. Dr Eva-Maria Sadowski, Museum f\u0026uuml;r Naturkunde, Berlin is thanked for searching for the original material of the species described by Caspary and Conwentz. We acknowledge the European Synchrotron Radiation Facility (ESRF) for provision of synchrotron radiation facilities, and we would like to thank Paul Tafforeau for assistance and support in using beamline BM18.We are grateful to Dr Hanne N. Rasmussen, Department of Geosciences and Natural Resource Management, University of Copenhagen, for her skilful reconstruction and drawing of Ilex wennebergii as it is supposed to have looked before fossilization.This study was supported by a grant from Danish Technological Institutes performance contract 2021-2024, entered with the Danish Agency for Higher Education and Science, under The Ministry of Higher Education and Science Denmark.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe fossil specimen is part of the amber collection at the Natural History Museum of Denmark and the Danekr\u0026aelig; fossil trove. All observations made during this study are included in this paper.\u003c/p\u003e\n\u003cp\u003eCorrespondence should be addressed to FNR.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYao, X., Song, Y., Yang, J-B., Tan, Y. \u0026amp; Corlett, R. Phylogeny and biogeography of the hollies (\u003cem\u003eIlex\u003c/em\u003e L., Aquifoliaceae). \u003cem\u003ePlant. Syst. Evol.\u003c/em\u003e \u003cb\u003e59\u003c/b\u003e, 73\u0026ndash;82 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoizeau, P-A., Savolainen, V., Andrews, S., Barriera, G. \u0026amp; Spichiger, R. Aquifoliaceae. In The Families and Genera of Vascular Plants XIV, Flowering Plants, Eudicots (ed Kubitzski, K.) 31\u0026ndash;36 (Springer, (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoizeau, P. A., Barriera, G., Manen, J. F. \u0026amp; Broennimann, O. Towards an understanding of the distribution of \u003cem\u003eIlex\u003c/em\u003e L. (Aquifoliaceae) on a World-wide scale. \u003cem\u003eBiologiske Skrifter\u003c/em\u003e. \u003cb\u003e55\u003c/b\u003e, 501\u0026ndash;520 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKnobloch, E. \u0026amp; Mai, D. H. 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Eocene vegetation patterns reconstructed from plant.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ediversity \u0026mdash; A global perspective. \u003cem\u003ePalaeogeography, Palaeoclimatology, Palaeoecology\u003c/em\u003e 247, 243\u0026ndash;271 (2007).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4958893/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4958893/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe genus \u003cem\u003eIlex\u003c/em\u003e (holly) in the monotypic family Aquifoliaceae contains more than 600 species distributed worldwide. Pollen fossils and macrofossils of fruits of \u003cem\u003eIlex\u003c/em\u003e are known from the late Cretaceous (90 Ma) and verified leaves from upper Miocene (10 Ma), but until now only a few more or less incomplete flower fossils have been suggested as referable to this genus.\u003c/p\u003e \u003cp\u003eThis paper reviews the fossil flowers earlier attributed to \u003cem\u003eIlex\u003c/em\u003e and presents a complete and indisputable staminate \u003cem\u003eIlex\u003c/em\u003e flower from Baltic amber (34\u0026ndash;38 Ma) examined in fine detail with Synchrotron X-ray tomography. This find may challenge the hypothesis of a late Neogene dispersal of the genus from a center of origin in subtropical East Asia.\u003c/p\u003e","manuscriptTitle":"First indisputable fossil Ilex (Aquifoliales: Aquifoliaceae) flower found in Baltic amber","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-07 12:57:25","doi":"10.21203/rs.3.rs-4958893/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-09T04:19:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-01T01:03:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-26T02:52:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"182264107211796617581094652360569439204","date":"2024-09-24T01:12:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"183903650666769716964907371863783093171","date":"2024-09-21T04:13:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-20T15:31:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-20T15:28:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-09-05T18:23:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-04T12:17:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-08-22T14:46:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3f4adbfb-8bb9-42bd-852a-9fd6e90c4103","owner":[],"postedDate":"October 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":38392645,"name":"Biological sciences/Evolution"},{"id":38392646,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2024-11-18T19:27:20+00:00","versionOfRecord":{"articleIdentity":"rs-4958893","link":"https://doi.org/10.1038/s41598-024-78892-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-11-12 15:58:05","publishedOnDateReadable":"November 12th, 2024"},"versionCreatedAt":"2024-10-07 12:57:25","video":"","vorDoi":"10.1038/s41598-024-78892-4","vorDoiUrl":"https://doi.org/10.1038/s41598-024-78892-4","workflowStages":[]},"version":"v1","identity":"rs-4958893","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4958893","identity":"rs-4958893","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}