# 100-million-year-old conifer tissues from the mid-Cretaceous amber of Charente (western France) revealed by synchrotron microtomography

Jean-David Moreau, Didier Néraveau, Vincent Perrichot, Paul Tafforeau

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Jean-David Moreau, Didier Néraveau, Vincent Perrichot, Paul Tafforeau. 100-million-year-old conifer tissues from the mid-Cretaceous amber of Charente (western France) revealed by synchrotron microtomography. Annals of Botany, 2017, 119 (1), pp.117-128. 10.1093/aob/mcw225. insu-01415770

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# 100-million-year-old conifer tissues from the mid-Cretaceous amber of Charente (western France) revealed by synchrotron microtomography

Jean-David Moreau¹*, Didier Néraveau¹, Vincent Perrichot¹ and Paul Tafforeau²

¹CNRS UMR 6118 Géosciences Rennes, Université de Rennes 1, Campus de Beaulieu bât. 15, 263 Avenue du Général Leclerc, 35042 Rennes, France and ²European Synchrotron Radiation Facility, 71 avenue des Martyrs, 38000 Grenoble, France

*For correspondence. E-mail jean.david.moreau@gmail.com

Received: 8 July 2016 Returned for revision: 8 August 2016 Accepted: 15 September 2016 Published electronically: 10 December 2016

* **Background and Aims** Terrestrial plant remains in fossilized tree resin are relatively common. However, histology and preservation of plants entombed in Cretaceous ambers remain poorly known. We report an exquisitely preserved conifer leafy axis from 100-million-year-old opaque amber of western France that is assignable to *Glenrosa carentonensis* Moreau, Néraveau, Tafforeau & Dépré. The purpose of this paper is to discuss the taphonomy and the use of microtomography for studies of palaeobotanical remains in amber.

* **Methods** A leafy axis was examined using propagation phase-contrast X-ray synchrotron microtomography with voxel sizes of 14-9, 1-4, and 0-7 µm.

* **Key Results** The conifer leafy axis described is preserved in three dimensions. Despite desiccation of the specimen within the surrounding amber, the cuticle, as well as most of inner tissues, is preserved in three dimensions down to the cellular level. Epidermis, palisade parenchyma, spongy parenchyma, transfusion tracheids and vascular bundles are clearly distinguished.

* **Conclusions** Gross morphology and histology of the specimen were revealed using synchrotron microtomography, allowing an unprecedented resolution for the study of soft-bodied plants entombed in amber. The study reveals a peculiar combination of authigenic and duripartic preservation as well as permineralization, and highlights the complexity of taphonomic processes that can occur in amber inclusions. This fossil demonstrates the difficulty of studying amber-preserved plant remains under certain conditions.

**Key words:** Amber, Cretaceous, conifers, *Glenrosa carentonensis*, histology, France, X-ray synchrotron microtomography.

## INTRODUCTION

Preservation of terrestrial plant remains in amber, a fossilized tree resin, is relatively common and has often provided excellent examples. Fossils of various angiosperms, bryophytes, ferns and gymnosperms have been reported from many amber deposits worldwide, but detailed descriptions have focused mostly on inclusions from Miocene Dominican and Mexican ambers (Miranda, 1963; Calvillo-Canadell et al., 2010; Chambers et al., 2011; Heinrichs et al., 2014, 2015a, b; Lee et al., 2015; Schneider et al., 2015; Poinar and Struwe, 2016), Palaeogene European ambers (Frahm, 1996; Grolle and Meister, 2004; Koller et al., 2005; Dörfelt and Schmidt, 2007; Schmidt and Dörfelt, 2007; Ignatov and Perkovsky, 2011; Heinrichs et al., 2015a, b; Mamontov et al., 2015; Sadowski et al., 2015, 2016), and Cretaceous Burmese and New Jersey ambers (Crepet and Nikon, 1998; Poinar and Buckley, 2008; Hentschel et al., 2009; Chambers et al., 2010; Heinrichs et al., 2012; Schneider et al., 2016). Pollen grains, sporangia, sporo-phytes, leaves and flowers can be very finely preserved in amber, allowing detailed morphological studies; however, analyses with standard optical microscopes are limited to the gross morphology of the amber inclusions, and only the use of invasive techniques such as amber thin-sectioning has permitted access to cellular and histological details of the plants (Koller

et al., 2005). It is only recently that non-invasive methodologies in computerized tomography (CT) scanning have been applied to determine the detailed structural features of a small fossil flower preserved in Cretaceous Burmese amber (Crepet et al., 2016).

Here, we describe the gross morphology and histology of an exquisitely preserved conifer leafy axis from mid-Cretaceous (early Cenomanian) amber of western France, using non-invasive propagation phase contrast X-ray synchrotron microtomography (PPC-SRµCT; Tafforeau et al., 2006; Woen et al., 2006). This specimen was discovered in 2005 in a block of opaque amber during a large radiographic survey performed using propagation phase contrast to detect inclusions in such amber (Lak et al., 2008). Because the histology and preservation of plants entombed in Cretaceous ambers remain poorly known, the purpose of this paper is to discuss the taphonomy and use of microtomography for studies of palaeobotanical remains in amber.

## GEOLOGICAL SETTING

The amber was collected from a locality named La Buzinie, in the Champniers area, 6 km north of Angoulême (Charentes region, western France). Four amber-bearing beds, all early
Cenomanian in age, were discovered at this locality that was accessible only temporarily during road works in 2004 (Perrichot *et al.*, 2007). Cenomanian deposits are regionally divided into seven lithological units named A–G, the lower Cenomanian including four subunits A2, B1, B2 and B3 (Néraveau and Moreau, 1989). At La Buzinie, amber has been found in these four subunits but the main accumulations were observed in A2 and B2 (Perrichot *et al.*, 2007). Subunit A2 consists of a thin and discontinuous lignitic deposit resting unconformably on the calcareous Jurassic substratum. Compression fossil plants with preserved cuticle were reported together with amber from subunit A2. Subunit B1 consists of a grey, beige or pink bioclastic limestone yielding foraminifers such as *Orbitolina concava*, and containing at its base amber nodules reworked from the A2 lignites. Subunit B2 consists of glauconitic green sand and sandstones locally showing black lignitic lenses in its lower midpart. Subunit B3 consists of limestones with lignitic sand lenses. The fragment of amber studied herein was collected from subunit A2, which has already yielded diverse arthropod and micro-organism inclusions (Perrichot *et al.*, 2007; Lak *et al.*, 2008; Vršanský, 2008; Edgecombe *et al.*, 2009; Lak and Nel, 2009; Girard, 2010).

## MATERIALS AND METHODS

Indeterminate plant remains have been observed in several mid-Cretaceous Charentese ambers, corresponding to fragments of wood or leaf axes. Most come from translucent and opaque ambers with arthropods from the localities of Archingeay-Les Nouillers or Fouras, in Charente-Maritime (Perrichot, 2004; and our pers. obs.). This paper presents a unique leafy axis embedded in an opaque fragment of milky amber (Figs 1–3) from La Buzinie, in Charente (Perrichot *et al.*, 2007). The leafy axis is ascribed to the genus *Glenrosa* Watson & Fisher, an extinct Cretaceous conifer. This plant is characterized by an unusual stomatal arrangement inside crypts (Watson and Fisher, 1984). The specimen studied herein constitutes the first instance of *Glenrosa* remains included in fossil resin. The leafy axis is exquisitely preserved in three dimensions, showing details of the cuticle and inner microstructures (Figs 1–7). Despite slight desiccation of the specimen within its surrounding amber, an external cast of the original plant is preserved. Because this fossil plant is unique and inaccessible using traditional non-destructive techniques (e.g. light microscopy), we used a 3D imaging approach. The specimen was originally discovered during a large radiographic survey of opaque amber blocks performed on the ID19 beamline (European Synchrotron Radiation Facility, Grenoble, France; Tafforeau *et al.*, 2006) using propagation phase contrast in 2005, with a voxel size of 5·06 µm and 1-m sample–detector distance. Amber blocks were glued to plastic plates and placed in water to remove general absorption contrast and structures coming from the surface, in order to increase visibility of the inclusions inside the blocks (Lak *et al.*, 2008). The fragment of amber was later examined using PPC-SRµCT. Experiments were conducted at the ID19 beamline. The scans of the specimen were performed using an effective energy of 25–30 keV, and voxel sizes of 14·9, 1·4 and 0·7 µm. We used a filtered beam monochromatized by an Si-111 double-crystal

monochromator or by a single-bounce multilayer (Paganin *et al.*, 2002; Sanchez *et al.*, 2012). Details of the acquisition parameters are given in Table 1. The data were reconstructed using a filtered-backprojection algorithm (ESRF software PyHST2, Mirone *et al.*, 2014), coupled with a modified single distance phase retrieval procedure (Paganin *et al.*, 2002; Sanchez *et al.*, 2012). Residual ring artefacts were corrected on reconstructed slices using inhouse MATLAB code (Lyckegaard *et al.*, 2011). The 3D reconstructions were achieved with the software VG Studio Max, versions 2.0 and 2.2 (Volume Graphics, Heidelberg, Germany). The virtual dissection and segmentation were achieved using 3D region growing and manual selections. The specimen is housed at the Geological Department and Museum of the University of Rennes 1, under collection number IGR.BUZ-7. The 3D data presented in this article, as well as segmentations and additional images, are available online at http://paleo.esrf.eu.

## SYSTEMATIC PALAEONTOLOGY

Order — Coniferales

Family — *Incertae sedis*

Genus — *Glenrosa* Watson & Fisher, 1984 emend. Srinivasan, 1992

Species — *Glenrosa carentonensis* Moreau, Néraveau, Tafforeau & Dépré, 2015

*Material.* Amber piece IGR.BUZ-7 from the road works of La Buzinie, Champniers, Charente, SW France; scan ESRF no. LB-015 (Figs 1–7).

*Description.* Leafy axis unbranched, up to 14·5 mm long and 3·3 mm wide. Leaves helically arranged, arising at up to 70° and showing a phyllotaxy 8/21 (Figs 1–3). Leaves triangular in abaxial and adaxial views, rhomboidal in transversal section, claw-shaped in sagittal section and well-keeled on both sides; up to 2·7 mm long and up to 1·5 mm wide, with margin entire and apex obtuse to acute (Figs 1–3). Cuticle 4–16 µm thick (Figs 4, 5A, B and 7A, B), its outer surface with well-marked outline of epidermal cells (Fig. 4); each surface with randomly distributed stomatal crypts (Figs 4 and 7B). Crypts lightly deeper than wide. Stomatal crypts 50–100 µm in diameter, 95–100 µm deep. Crypt apertures oval, 30–80 µm in diameter, and surrounded by up to 11 epidermal cells. Crypts bearing elongated papillae with acute apices. Stomata located at the bottom of the crypt, up to 55 µm long and 40 µm wide. Epidermis a single layer 8–25 µm thick (Figs 4A, B and 7A, C). Ordinary epidermal cells polygonal, square to rectangular, 14–35 µm long and 11–30 µm wide. Anticlinal walls of ordinary cells straight, up to 4 µm thick. A thin and single layer of elongated hypodermal cells locally preserved on adaxial surface (Fig. 7D). Hypodermis absent from the abaxial surface. Palisade parenchyma a single, 35–100-µm-thick layer of cells occurring along the entire length and on both sides of the leaves (Figs 5 and 6). Cells elongated, 35–100 µm long and 11–40 µm wide. Spongy parenchyma of up to seven layers of 80-µm-wide rounded cells (Figs 5, 6 and 7E). Transfusion tracheids only partially preserved, present on abaxial side. Cells ovoid to elongated, up to 90 µm long. Cell walls convex to straight, showing numerous
<figure><img src="image_2.png" /></figure>

FIG. 1. PPC-SRµCT, virtual section of *Glenrosa carentonensis*, specimen IGR.BUZ-7. (A) Longitudinal section of the leafy axis showing helically arranged leaves. (B,C) Transversal sections of the leafy axis. Abbreviations: Cu., cuticle; Ex., external cast of the plant remains; Ti., tissues. Scale bars = 2 mm. Voxel size, 14-92 µm.

<figure><img src="image_3.png" /></figure>

FIG. 2. PPC-SRµCT, 3D renderings of *Glenrosa carentonensis*, specimen IGR.BUZ-7. Virtual extraction of the leafy axis, with opaque resin progressively eroded from left to right. Scale bars = 10 mm. Voxel size, 14-92 µm.
<figure><img src="image_4.png" /><figcaption>FIG. 3. PPC-SRµCT, 3D renderings of the leafy axis of *Glenrosa carentonensis*, specimen IGR.BUZ-7. (A–C) Lateral view; (D–F) top view; (G–I) bottom view. From left to right, external cast of the leafy axis (A, D, G) is progressively and virtually eroded in order to expose only the cuticle (C, F, I). Scale bars = A–C, 5 mm; D–I, 2 mm. Voxel size, 14-92 µm.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>FIG. 4. PPC-SRµCT, 3D renderings of the cuticle of *Glenrosa carentonensis*, specimen IGR.BUZ-7. (A) External view displaying apertures of stomatal crypts and well-marked outline of polygonal epidermal cells. (B) Internal view displaying apertures of stomatal crypts (S.c) and epidermal cells. Scale bars = 100 µm. Voxel size, 0.7 µm.</figcaption></figure>

pits. A single vascular bundle present inside leaves (Figs 5 and 6B–D). Vascular bundle ending before the leaf apex. Leafy axis and vascular bundle form an angle of up to 45°.

*Remarks.* All measurements were directly taken on the cuticles and tissues, not on the external cast of the leafy axis. Because of limited resolution of the tomographic data, tissues inside the vascular bundle cannot be identified but they are probably locally preserved. The genus *Glenrosa* is represented by seven species that are late Barremian to Cenomanian in age (Watson and Fisher, 1984; Srinivasan, 1992; Zhou *et al.*, 2000; Gomez *et al.*, 2012; Moreau *et al.*, 2015). *Glenrosa carentonensis* differs greatly from other species of the genus in developing a phyllotaxy 8/21, claw-shaped leaves, a thicker cuticle, and a

higher number of papillae and stomata per crypt. The leaves, cuticles and tissues of the present specimen from Champniers are only slightly smaller than specimens of *Glenrosa carentonensis* described from the silicified Cenomanian deposits of the Font-de-Benon quarry between Archingeay and Les Nouillers, Charente-Maritime (Moreau *et al.*, 2015).

## DISCUSSION

### Exceptional plant preservation

Abundant and diverse plant meso- and mega-remains have been reported from the Cretaceous clayey and lignitic deposits
<figure><img src="image_6.png" /><figcaption>FIG. 5. PPC-SRµCT, virtual 2D histological sections of *Glenrosa carentonensis*, specimen IGR.BUZ-7. (A) Transversal section of leafy axis showing cuticle and tissues. (B) Tangential section of a leaf, showing cuticle and tissues. (C) Detail of tissues including the spongy parenchyma, the transfusion tracheids and the vascular bundle. (D) Detail of tissues including the palisade parenchyma, the spongy parenchyma, the transfusion tracheids and the vascular bundle. Abbreviations: P.Pa., palisade parenchyma; S.Pa., spongy parenchyma; Tr., transfusion tracheids; V.B., vascular bundle. Scale bars = 200 µm. Voxel size, 0.7 µm.</figcaption></figure>

of western France, mainly from the late Early Cretaceous (Albian) and the early Late Cretaceous (Cenomanian) of Charente-Maritime (Néraseau *et al.*, 2002; Gomez *et al.*, 2004, 2008; Coiffard *et al.*, 2009), Dordogne (Saint-Martin *et al.*, 2013) and Maine-et-Loire (Néraseau *et al.*, 2013). Due to compaction of sediments, these remains are highly compressed. They commonly consist of impressions and compressions with or without cuticles. Impressions represent the weakest preservation. Fossil plants are limited to external casts; only the gross morphology can be discussed. In the most common case, Cretaceous lignitic deposits from western France yield plant compressions with well-preserved cuticles (Gomez *et al.*, 2004, 2008). Cuticles may show casts of the most peripheral epidermal cells, allowing the description of microstructural details such as morphology and size of ordinary cells and stromal apparatuses. However, these compressions with cuticles

do not preserve any histology. By contrast with impressions and compressions, the conifer leafy axis described in the present paper is preserved in three dimensions. Moreover, the inclusion shows exquisitely preserved gross morphology, cuticle and most of the inner tissues, including epidermis, palisade parenchyma, spongy parenchyma, transfusion tracheids and vascular bundle.

Plant meso-remains preserved inside amber generally vary from isolated cuticle fragments (Schmidt *et al.*, 2010) to internal empty cavities with or without collapsed tissues (Schopf, 1975; Rikkinen and Poinar, 2000), or plants with well-preserved histology (Koller *et al.*, 2005). In Tertiary amber, some specimens show preservation of tissues in three dimensions. Palisade parenchyma of angiosperm leaves and most tissues and organelles in angiosperm leaflets have been described from Tertiary Dominican amber (Grimaldi *et al.*, 1994; Poinar
<figure><img src="image_7.png" /><figcaption>FIG. 6. PPC-SRµCT, 3D reconstruction of the histology of one leaf of *Glenrosa carentonensis*, specimen IGR.BUZ-7. (A) External view of the palisade parenchyma. (B) Virtual 3D thick section in the tangential plane. (C) Virtual 3D thick section in the sagittal plane. (D) Virtual 3D thick section in the transversal plane. Abbreviations: P.Pa., palisade parenchyma; S.Pa., spongy parenchyma; Tr., transfusion tracheids; V.B., vascular bundle. Scale bars = 200 µm. Voxel size, 0.7 µm.</figcaption></figure>

et al., 1996). Koller et al. (2005) reported an exceptionally well-preserved cypress leafy axis from Eocene Baltic amber, showing tissues of the entire stem and leaves, their cells and organelles. Preserved organic cell contents of pollen grains included inside amber have been described from the Lower Eocene of the Paris Basin, in northern France (De Franceschi et al., 2000; Dejax et al., 2001).

In contrast, details of inner fossil plant histology have never previously been reported from Cretaceous amber. Rare studies reported microstructures such as stomatal apparatuses, although observation was limited to the external view of plants (Poinar et al., 2015). Only Crepet et al. (2016) recently mentioned tissue boundaries inside an Early Cretaceous flower. With the

conifer presented in this paper, we provide the most detailed description of an amber-preserved plant inclusion so far. Except inside amber, fossil plants preserving inner tissues are exceedingly rare in Cretaceous deposits of western France. The only other kind of plant preservation providing histological information consists of silicified permineralizations/petrifications (Moreau et al., 2014a, b, 2015).

Taphonomic insights

This plant inclusion shows very uncommon preservation features. An external cast of the entire leafy axis is preserved in
<figure><img src="image_8.png" /><figcaption>FIG. 7. PPC-SRµCT, virtual 2D histological sections of *Glenrosa carentonensis*, specimen IGR.BUZ-7. (A) Detail of thick cuticle and epidermis. (B) Cuticle in transversal section showing deep stomatal crypts. (C) Cuticle in longitudinal section showing polygonal epidermal cells. (D) Epidermis in longitudinal section showing rows of elongated cells. (E) Detail of spongy parenchyma showing rounded cells. (F) Details of transfusion tracheids showing small pits. Abbreviations: C.Ep., cells of epidermis; P.Pa., palisade parenchyma; S.Cu., surface of cuticle; S.Pa., spongy parenchyma; Tr., transfusion tracheids; V.B., vascular bundle. Scale bars = 200 µm. Voxel size: A and E, 1.4 µm; B–D and F, 0.7 µm.</figcaption></figure>
<table><caption>TABLE 1. Acquisition parameters of the 3D imaging approach</caption><thead><tr><th>Monochromator/multilayer</th><th>Si-111 double crystal monochromator</th><th>Multilayer</th><th>Multilayer</th></tr></thead><tbody><tr><td>Voxel size (µm)</td><td>14.9</td><td>0.7</td><td>1.4</td></tr><tr><td>Energy (keV)</td><td>25</td><td>30</td><td>25</td></tr><tr><td>Propagation distance (cm)</td><td>7</td><td>2.0 – 4.3 – 22.6</td><td>10</td></tr><tr><td>Camera</td><td>FReLoN 2000 CCD</td><td>FReLoN 2000 CCD</td><td>FReLoN 2000 CCD</td></tr><tr><td>Scintillator</td><td>Gadox 10</td><td>Ce (YAG) 25</td><td>Ce (YAG) 25</td></tr><tr><td>Insertion devices-gap (mm)</td><td>W150_40</td><td>W150_40.0; U32_12.1; U44_15.8</td><td>W150_38; U32_12</td></tr><tr><td>Number of projections</td><td>1200</td><td>2500</td><td>1200</td></tr><tr><td>Exposure time (s)</td><td>0.2</td><td>1.5</td><td>0.3</td></tr></tbody></table>

three dimensions inside the resin. Cuticle is nearly totally detached from this cast, showing a smaller volume than the external imprint of the leafy axis (Figs 1–3 and 5A, B). Inner tissues are slightly retracted, not in contact with the cuticle (or only partially), occupying a smaller volume. Tomographic analyses show that preservation features of histology are not homogeneous. On tomographic reconstructions, the low-density structures consist of dark areas. In some areas the tissues show well-preserved anticlinal and periclinal walls, and empty intracellular spaces. By contrast, some parts of the plant histology show dense material inside intracellular spaces, mainly located in the most peripheral tissues (e.g. palisade parenchyma, spongy parenchyma). This dense material appears white on tomographic reconstructions and is denser than amber. Microcrystallizations of pyrite have often been observed from mid-Cretaceous amber from Charente-Maritime (Perrichot, 2005; Girard, 2010) and thus pyritization of intracellular spaces cannot be excluded. Pyrite has also been reported from Baltic amber (Garty et al., 1982; Kowalewska and Szwedo, 2009).

Although the leafy axis and the tissues are preserved in three dimensions, they are not preserved in their original volume. The entire structure is slightly shrunken. It does reflect merely unilateral mechanical flattening induced by diagenetic compaction. Here, the deformation is homogenous in the three dimensions of the space. All features show that the axis was not dehydrated before being engulfed in fresh resin. Several steps can be distinguished in the taphonomic processes, for which we propose the following hypothesis (Fig. 8):

1. The fresh leafy axis was embedded inside liquid resin.

2. The resin dried rapidly, forming an external cast of the plant.

3. The tissues were dehydrated. They originally contained a large quantity of water, in contrast to the cuticle which is hydrophobic, which could explain the differential retraction compared with that of the tissues. Retraction of inner tissues probably started early during diagenesis. However, the loss of water alone cannot explain the structural modifications. Strong molecular modifications should also have contributed to the morphological transformations. Biochemical studies on Tertiary resins and plants have shown that although the molecular composition of resin remains constant through diagenesis, those of the plant inclusions become extensively degraded (Stankiewicz et al., 1998).

4. Intracellular spaces of the most external tissues were locally microcrystallized. One of the characteristics of the Charentese amber is the presence of mineral crystallizations (e.g. pyrite) along the abundant microcracks, as well

as along the planes between resin laminae, and in the insect inclusions. In this context, an amber piece cannot be considered as a closed system: fluid infiltrations, transport of material from outside to inside, and precipitation of minerals become possible.

The preservation and classification of amber-preserved plant inclusions have long been debated. As explained by Schopf (1975), they constitute a peculiar way of preservation that must be distinguished from that of the resin itself. When preservation is limited to an external empty cast, Schopf (1975) suggested an 'authigenic preservation' that consists of surface replicas at the time of deposition or very early during diagenesis. When the plant itself is preserved, Schopf (1975) tentatively classed inclusions in 'coalified compressions'. He defined the latter as preservation with undistorted non-mineralized tissues becoming coal. Martín-Closas and Gomez (2004) classified amber inclusions as a particular kind of 'duripartic preservations'. They define the latter as preservation with weak morphoanatomical and molecular modifications of the original structures. Duripartic preservations are produced when the plant material is in balance with the mineral environment. However, as mentioned above, at the molecular scale, strong modifications have been reported for fossil plant tissue included inside resins (Stankiewicz et al., 1998).

Based on the morphological features observed here, the taphonomic processes involved in preservation of plant inclusions are extremely complex and can be multiphased. The leafy axis from Champniers amber provides an example of the difficulty involved in classifying amber-preserved plants. The specimen presented here combines features of 'authigenic preservations' (external moulding of the structure sensu Schopf, 1975), 'duripartic preservations' (exquisite preservation of the gross morphology and tissues with weak morphoanatomical modifications sensu Martín-Closas and Gomez, 2004) and 'permineralizations' (microcrystallizations inside intracellular spaces, by infiltration of minerals sensu Schopf, 1975).

### Synchrotron microtomography for the study of plant inclusions in amber

Plant inclusions inside translucent ambers are generally studied using light microscopy, but this standard, easy-to-use technique is not appropriate with dark, cloudy or opaque amber. Furthermore, observations of plant inclusions are commonly limited to gross morphology and outer microstructures while the histology, if preserved, remains rarely observed. On the
<figure><img src="image_9.png" /><figcaption>FIG. 8. Hypothetical steps explaining the plant preservation seen in amber piece IGR.BUZ-7. (A) Embedding of the leafy axis in fresh resin, including cuticle (Cu.) and tissues (Ft.). (B) After partial or complete drying of the resin, the early stages of plant dehydration produce a slight retraction of the entire structure, resulting in an external cast (Ex.) of the conifer inside the resin. (C) Because of the large quantity of water originally contained inside tissues, complete plant dehydration produces a differential retraction compared with the cuticle, which is hydrophobic. (D) After complete dehydration, intracellular spaces of the most external tissues are locally microcrystallized (Mi.). Z1., zone 1 corresponds to the cavity between the external cast of the plant remains and the cuticle; Z2, zone 2 corresponds to the cavity between cuticle and tissues.</figcaption></figure>

many described flowers from Mesozoic and Tertiary ambers, most of the inner microstructures and histology are inaccessible using standard techniques (Poinar, 2002; Poinar and Chambers, 2005; Santiago-Blay et al., 2005; Castañeda-Posadas and Cevallos-Ferriz, 2007; Calvillo-Canadell et al., 2010; Chambers et al., 2010, 2011; Poinar and Steeves, 2013; Poinar et al., 2013). Only rare cases using light microscopy have provided partial tissue details and cell information (Heinrichs and Schmidt, 2010). The few studies that have focused on inner structures used invasive techniques such as thin sectioning or manual breaks, combined with fluorescence or electron microscopy (Grimaldi et al., 1994; Koller et al., 2005). The interface between resin and tissues can be observed by opening the amber mechanically.

Cutting-edge methodologies such as CT scanning have only recently been used in the study of structural features of an amber-preserved flower (Crepet et al., 2016). PPC-SRµCT has also proved to be efficient in the field of palaeobotany. Beamlines from several synchrotrons have previously tested tomographic protocols on diverse kinds of plant preservations, such as impressions, compressions, charcoals, lignites, permineralizations and petrifications (Feist et al., 2005; Friis et al., 2007; Moreau, 2014; Moreau et al., 2014, 2015, a, b, c, Strullu-Derrien et al., 2014), allowing a description of the gross morphology and histology without a destructive approach. This technique enables the production of an infinite number of virtual palaeohistological sections. Cuticle and most of the outermost to innermost tissues can be characterized. Given that amber inclusions are one of the most informative kinds of plant preservation, this virtual 3D palaeohistological approach should provide new opportunities for palaeobotany.

## ACKNOWLEDGEMENTS

We thank Alain Couillard for providing the amber piece containing the specimen described here, and Malvina Lak for contributing to synchrotron imaging and for original discovery of the specimen. We thank George Mustoe and the anonymous reviewer who provided useful comments and suggestions. We thank Jean-Paul Saint-Martin who kindly shared bibliographic references with us. This work was supported by funding from Géosciences Rennes, CNRS-UMR6118, to J.-D.M., D.N. and V.P.; and the European Synchrotron Radiation Facility, to J.-D.M. and P.T. Partial support was provided by French National Research Agency grant BLAN07-1-184190 (project AMBRACE to D.N.).

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