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

doi:10.21203/rs.3.rs-4958893/v1

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

https://doi.org/10.21203/rs.3.rs-4958893/v1

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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

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.

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. 2D) was done from the light microscopy and CT images, CT-video clip (Supplementary video S3) and a 3D print from 3D dataset.

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. 1a, b, d, f) and corolla partially detached from the receptacle (Fig. 1c, 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. 2a). 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. 2b, 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. 2b-c). The flower is withered, the corolla is partly detached, and the anthers are collapsed (Fig. 1c, 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 2d 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).

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. wennebergii 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.

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).

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.

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No competing interests reported.

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

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