Introduction

Ilex contains more than 600 extant species and is the the most species-rich genus of dioecious woody plants1. The leaves of more than 60 species of Ilex are used for beverages and several species are known for their medicinal properties. The evergreen branches with red fruits of I. aquifolium and I. chinensis are widely used as symbols of Christmas and New Year in many parts of the world2.

Ilex is well represented in the fossil record as pollen3 as well as macrofossils, e.g., fruit stones4 and leaves5. However, only six fossil species have been discussed as possible Ilex-flowers, all inclusions in Baltic amber and published in the 19th century. Five of the six species were described and beautifully illustrated in colour by Conwentz in 18866, all of which were reproduced enlarged in black and white in Schenck in 18907.

Ilex prussica was mentioned informally by Caspary in 18728 and validated as I. prussica Casp. ex Conw. by Conwentz6 who added another fossil species, Ilex minuta Conw6. The illustrations show two tetramerous flowers with pedicel (pubescent in both species) and calyx (marginally pubescent in I. minuta). Ilex prussica is 5 mm high and I. minuta approximately 2.5 mm from the base of the flower to the apex of the stamens. The petals appear to be free but may be merged slightly at the base. The petals are upright, with slightly incurved apices, in I. prussica ovate, and in I. minuta spatulate. The stamens are significantly longer than the petals. The stamens appear to be opposite the petals, but the accompanying floral diagrams indicate that they alternate with the petals. A pistil or pistillodium cannot be seen. Conwentz’s contemporary colleague Loesener9 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. Schenck7,10 was also highly sceptical of the placement of these fossils in Ilex, but he did not suggest any other genera as possible extant relatives.

Three fossil detached corollas with epipetalous stamens were described in Ilex by Caspary11. Ilex aurita Casp. is hexamerous with unequal petal lobes. It was not depicted by Conwentz, who was in doubt about the determination. However, Loesener9 accepted this fossil as an Ilex because of its overall similarity to extant species. Ilex minor Casp. is also hexamerous and was transferred to Sambucus as S. succinea by Conwents6. Loesener9 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 subsequently transferred it to Sambucus multiloba (Casp.) Conw6. after having studied more material of what he considered the same species. Loesener suggested9 that it could be a teratological female Ilex flower because of the rather small anthers, which might be staminodia. However, Schenck7accepted 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 closely resembling Sambucus succinea was shown on the front page and on p. 33 as “Ilex sp.” by Bachofen-Echt12 in 1949, but without a scale or indication of collection or repository.

Celastrinanthium hauchecornei (Celastraceae) is a tiny cyme with three tetramerous unopen buds, described and depicted by Conwentz6. Loesener noted9 that it looked more like an Ilex, but decisive identification was not possible. It was accepted in Celastraceae by Schenck7.

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 II13. 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.

Materials and methods

The material studied is a piece of Baltic amber, approximately 80 × 70 × 15 mm (Fig. 1), found at the west coast of Jutland, Denmark, near Hvide Sande, by the Danish amber enthusiast and collector Lars Wenneberg. Substantial amounts of Baltic amber are found along the shores of Denmark and are assumed to have been redeposited during the quaternary ice age14. 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. The flower is embedded with the underside near the surface of the piece, but the upper side, with details critical for identification, is covered by 12 mm amber and partly obscured by cracks and bubbles. It is labelled NHMD 872,634 at the Natural History Museum of Denmark and is nr. DK 1073 in the Danish “Danekræ” fossil trove15, disallowing destructive methods of investigation.

Fig. 1
figure 1

The amber piece NHMD 872634 at the Natural History Museum of Denmark, DK 1073 in the Danish “Danekræ” fossil trove. The red arrow points to the Ilex wennebergii flower. The red squares are positions of the six arthropod syninclusions, not treated in the present paper. 1: Beetle, 2: Ichneumonid wasp, 3: Fly, 4: Polyxenid millipede, 5: Mite, 6: Stalk-eyed fly.

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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 microcomputed 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 2 μm. 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. Subdatasets of each stamen were added and separately coloured to enhance visualisations. See Supplementary methods S1 for synchrotron scanning and 3D image processing details.

The reconstructed flower (Fig. 3d) was drawn from the light microscopy and CT images, a CT-video clip (Supplementary video S2) and a 3D print from the 3D dataset.

Fig. 2
figure 2

Ilex wennebergii sp. nov. NHMD 872634. (a) Light microscopy view from below through c. 2 mm amber, (b) view from above through c. 12 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.

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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 Ma16,17.

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 the extant European and Macaronesian species Ilex aquifolia L., I. colchica Pojark., I. perardo Aiton, and I. canariensis Poir. but about half the size of these species. The ratio filament: anther is 1:1, while in the extant species mentioned it is 3:1. Differs from the fossil alleged Ilex species 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. 2b, d, f) and corolla partially detached from the receptacle (Fig. 2c, 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 (Figs. 2a and 3a). 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, approximately 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. 3b, c).

Fig. 3
figure 3

Ilex wennebergii sp. nov. NHMD 872634.  (a-d) synchrotron CT scans, colouring as in Fig. 2, (a) view of flower fossil from below, (b) cross section and (c) longisection 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.

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Remarks

Fossils embedded in amber are usually impressions, i.e., empty voids containing no or very little organic material14. In this specimen the sepals, three of the four petals, the stamens and the carpels of the pistillodium are voids with scattered grains of degraded organic material. Only the pistillodium interior and one petal appear to be more solid, filled with a grainy substance (Fig. 3b-c). The flower is withered, the corolla is partly detached, and the anthers are collapsed (Fig. 2c, d, e, Supplementary Video S2). 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 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 bubble (c. 0.5 mm broad) is attached to the side and top of the pistillodium. Figure 3d shows 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) (Fig. 1).

Discussion

The present authors agree with Loesener9 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 hauchecornei6 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.

The fossils Ilex aurita, I. minor (= Sambucus succinea), and I. multiloba (= Sambucus multiloba) are just floral fragments. Without calyx and pistillodium or pistil 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 fossils described by Conwentz4 and Caspary11 have not been critically assessed since the comments by Loesener9 and Schenck7,10 in 1888 and 1890 despite the considerable interest in the phylogeny and palaeogeography of Ilex. Loesener later monographed the genus18,19, but did not comment further on the alleged Ilex fossils. The names of the species are listed in some textbooks and catalogues of plant fossils20,21,22,23, but Conwentz6 is just mentioned en passant in one modern paper on the history of Ilex24.

The cosmopolitan but very unequal extant distribution and the good fossil record, although without verifiable floral specimens, has made the genus Ilex a favourite object for the study of how clades speciate, spread and establish distributional areas1,3,24,25,26,27. However, recent estimates of the evolutionary chronology and views of Ilex palaeogeography rely mostly on the distinct pollen fossils5,28 and molecular clock calibrations of Ilex on a fruit stone from the Cretaceous and Miocene leaf fossils1,26. 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. Australia29), 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 drying1.

The genus Ilex is remarkable for its highly uniform flower morphology2, 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 diversification1. 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 and al1. 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 similar1,16 and this humid, warm-temperate to subtropical forest has covered large parts of the northern hemisphere in the Eocene30,31. The possibillity that Ilex dispersed from subtropical Asia already in the Eocene and that species found in Baltic amber are members of the crown clade of Ilex should not be disregarded.

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.