traditional and new microscopy techniques applied to the study of

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Traditional and new microscopy techniques applied to the study of microscopic fungi included in amber

M. Speranza1*, J. Wierzchos1, J. Alonso2, L. Bettucci3, A. Martín-González4, and C. Ascaso1 1IRN-CCMA, Department of Systems Ecology, CSIC, Serrano 115 bis, 28006, Madrid, Spain.2Museum of Natural Science Álava, Siervas de Jesús, 24, 01001 Vitoria-Gasteiz, Spain.3Mycology Department, Fac. of Science, University of the Republic, Julio H. Reissig 565, 11300, Montevideo, Uruguay.4Microbiology Department III, Faculty of Biology, Complutense University of Madrid, José A. Novais 2, 28040, Spain.

This review describes the classical and new microscopy techniques used for the study of fungi included in amber. Themain advances in this field regarding the study of highly fossiliferous amber deposits of Lower Cretaceous, dated 115-120Ma old, from Álava and Teruel (Spain) are presented. New approaches using methods as scanning electron microscopy inbackscattered electron mode, with energy dispersive X-ray spectroscopy microanalysis, at low temperature andtransmission electron microscopy were presented. These techniques give images with exceptional high magnification andresolution as well as important chemical and topographical information of microscopic fungi included in amber. Moreover,confocal laser scanning microscopy allow to determine the spatial relationship within microcenosis and offers a novelopportunity for in situ study of amber microorganisms preservation forms and mineralization processes. Fluorescencemicroscopy has been also successfully applied for detecting fungal autofluorescence in amber. The use of this microscopytechniques have opened the way to study microcenosis included in amber.

Keywords: amber; confocal laser scanning microscopy; fluorescence microscopy; fungi; fossil; light microscopy; lowtemperature scanning electron microscopy; scanning electron microscopy; scanning electron microscopy in backscatteredelectron mode; scanning electron microscopy in secondary electron; transmission electron microscopy; x-raymicroanalysis.

1. Introduction

First it is necessary to explain the scientific relevance of Spanish amber. This will help us to understand the uniqueopportunity offered by amber microinclusion in the study of ancient fungi and the obtention of palaeoecologicalinformation. Amber is a fossilized resin produced by the trunk and roots of certain trees, recovered from sediments that

have been preserved for hundreds of millions of years. Amber deposits are considered to be a specific type of fossilbioaccumulation that preserves exceptionally well palaeobiological and palaeoenvironmental information from the past.In Spain more than 100 localities with amber outcrops have been reported, and are located in the north east of theIberian Peninsula along the coastal line that existed during the Early Cretaceous and that are Albian in age [1, 2].However, until now amber with bioinclusions has only been reported in seven localities such as Peñacerrada andSalinillas (Álava Province), San Just and Arroyo de la Pascueta (Teruel Province) and recently in El Soplao (CantabriaProvince) [1, 4]. Early Cretaceous ambers with fossil inclusions are scarce, however such Spanish outcrops are highlyfossiliferous consequently which make them of great scientific interest [1]. In general amber is composed by complexmixtures of terpenes that include components that polymerize when exposed to light or oxygen [5]. Some palynologicaland chemical evidence has shown that the Spanish amber was produced by Araucariaceae (possibly from the genus Agathis) in the coniferous forests which grew in the north of the Iberian Plate about 115-121 Ma ago [1, 2, 6]. Spanishamber varies greatly in form and colour, ranging from yellow, red to dark brown, and from transparent, semitransparentor opaque, Fig. 1. Even unusually blue amber has recently been discovered in El Soplao outcrop [4]. These

macroscopically properties could be attributed to difference in tree sources, origin resin flows in the plants or chemicalmicroenvironments during resin secretion [3].

Fungi is one of the most diverse groups of organisms on Earth, approximately 1.5 million species have beenestimated, of which approximately 70.000 have been described [7]. The study of fossil fungi provides valuableinformation about diversity, structure and evolution of this group of microorganisms [8,9]. These studies have revealedthe role play by fungi in the establishment of land plants, in lignin degradation in Devonic forests and the parasiticrelationship with plants and animals [10]. However fungal fossils are scarce in geological records because the poorpreservation of the fungal structure [11].

Amber is a superb medium for the preservation of fragile organisms even their most delicate structures. This factexplains in part why so many taxa of vertebrates, invertebrates (especially arthropods) and plant inclusions in amberhave been reported [12]. However, in spite of the importance of microorganisms in cell evolution and the history of theEarth, fossil resins have not been extensively used as a data source for Paleomicrobiology studies. Studies related tofungi found in amber are scarce and their have been carried out in fossilized resin from diverse origin from Germany,

Birmania, Dominican Republic, France and recently from Ethiopia [13- 16]. The methodological limitation of themicroscopic inclusion analysis explains in part the scarce studies carried out for the detection, characterization and

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identification of microorganisms in amber. In previous works, various microscopy protocols were optimised for ambersamples treatment to study microorganisms included in this solid resin [17, 19]. The complexity of ambermicroinclusion analyses was resolve by using different microscopy strategies. That allowed us to obtain, at the sametime, structural, ultrastructural and chemical information about fungi and their preservation processes [17-21]. Here wesummarized some of these strategies with the aim of extend in their use in the study of microorganisms included inamber.

Fig. 1 Some examples of Spanish amber. a: Álava yellow amber from Peñacerrada; b: stalactite-shaped, c and d: amber specimensfrom San Just outcrop in Teruel. Bars= a: 25mm, b-d: 5 mm.

2. Samples sources and pretreatments

Several highly fossiliferous amber samples from Peñacerrada and Salinillas deposits, also know as Álava amber, wereinvestigated by our group. The methodology used for Álava amber extraction from outcrops and strategies for screening

biological inclusion in the laboratory were previously described [2, 22]. The Peñacerrada amber samples are housed inthe “Museo de Ciencias Naturales de Álava” (MCNA collection). Amber samples from Arroyo de la Pascueta and SantJust from Teruel outcrops were also analysed. These samples are housed in the “Fundación Conjunto Paleontológico deTeruel-Dinopólis” (CTP collection). For methodology used in the paleontological excavations for Teruel amberextraction see Delclòs et al. 2007 and Peñalver et al. 2007 [1, 3]. Arthropods are the predominant class of amber faunafound in Spanish outcrops, some fungi associated with insects were also analysed from Álava amber collection [2]. Inmost cases, amber samples were trimmed and polished manually to minimize light scattering for optimal microscopicobservation. The ground and polished amber pieces with inclusions were embedded in an epoxy resin (Epotek 301) toeliminate the ‘‘mirror-effect’’ of internal cracks when illuminated, according to the method previously described bySchlee and Dietrich [23] and used for Cretaceous ambers. This treatment also guarantees the conservation of amberprone to natural oxidation and eventual darkening and breakage.

3. Light and fluorescence microscopy

Light microscopy (LM) in bright field mode has been widely applied for investigating microorganisms in amber. Therefractive properties of amber have made it difficult to examine microorganisms such as fungi using LM. Furthermore,only when relevant taxonomic characters are preserved, such as reproductive structures and/or hyphal characteristics isit is possible to assign these fossil fungi to present-day groups. As consequence, and despite the great fungal diversity,only some genera and species of fossil fungi have been described [8, 11-16, 24-32]. In this sense, reports of fungiincluded in Spanish amber are also scarce [1, 3, 17-20, 33,34]. Fossilized mycelium and reproductive fungal structureshave been observed in Salinillas, Peñacerrada and San Just amber samples using LM, Fig. 2. Some of them are similarto that those observed in present-day zygomycetes, imperfect fungi and basidiomycetes (see text in Fig. 2). However,sometimes the low resolution of light microscopy cannot provide detailed resolution for the analysis of specific fungalstructures that are relevant for taxonomic studies.

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Fig. 2 Light microscope images of selected fungal structures founded in Spanish ambers from Peñacerrada (a, c and e), San Just (band d) and La Pascueta (f) outcrops. a: conidiophores with pyriform and smooth phragmoconidio (arrows), two-celled, similar to thatpresent in the actual genus Trichocladium, that includes dematiaceous species of hyphomycetes that occurring on wood in terrestrialor wood submerged in freshwater habitats; b: mature zygospore that resembles of that present in actual zygomycetes; c: fine hyphaewith a simple phialide (arrow) with aggregated conidia (head arrow) at the apex, similar to that observed in actual Acremonium genus, hyphomycetes conidial fungi; d: fungal hyphae forming arthroconidia (arrow) similar to that produced by species of the actualgenus Geotrichum; e: basidia typically four spored (head arrows) sterigmata (arrow); f: layer of fungal hyphae aggregates form a flatof fertile conidiophores (arrows). Bars= a: 20 µm, b: 50 µm, c and d: 10 µm, e: 20 µm.

Fluorescence microscopy (FM) is an important method in Mycology. Many pathogenic and saprophytic fungifluoresce under ultraviolet light and various species could be differentiated by their autofluorescence. Theautofluorescence is localized mainly in the cell wall and fungal hyphae septum (see section 6). In our case, FMexamination of the amber samples improves the visualization of fungal structures associated with insect, Fig. 3c.

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Fig. 3 Fungal mycelium associated with insect included in Álava amber. a: Thysanoptera overgrown by fungal mycelium observedby LM; b: bright field projection image of detail of the fringed wings with associated fungal hyphae (white arrows) and sporangium(red arrow); c: Fluorescence microscopy projection image (ext/em: 365/420-470 nm) of fungal hyphae autofluorescence (blackarrows) of the same zone observed in b; d: CLSM images of fungal hyphae with terminal sporangium (yellow arrow) localize inamber insect mould; (i), bright field image; (ii) strong autofluorescence (green) signal (ext/em: 488/515-545 nm) proceeded frommummified (non mineralized) outer part of hyphae; (iii) reflected laser light (red) from mineralized hyphae core and sporangium(yellow arrow); e: CLSM bright field image of fungal hyphae (black arrows) with terminal sporangium (yellow arrow) and f:

reflected laser light from mineralized (red) hyphae core (white arrows) and sporangium (yellow arrow). Bars= a: 100 µm, b and c:20µm, d: 20 µm, e and f: 15µm.

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In fossil records it is extremely difficult that the ecological interrelationships between organisms can be preserved.For this reason the interrelationships between fungi and animals are poorly documented. Mutualism, saprophitism andparasitism between insects and fungi have played a key role in their evolution [13,35,36]. Some of these relationshipsbetween fungi and insects have been observed in amber [11,13-15,25,30,31,35]. In a Thysanoptera specimen includedin Álava amber a vegetative mycelium and reproductive fungal structures, similar of that present in actual zygomycetesfungi, were observed by LM but improving when FM were used, Fig. 3 c.

4. Scanning electron microscopy in secondary and backscattered electron mode, and

energy dispersive X-ray spectroscopy microanalysis

One of the inherent limitations of light and fluorescence microscopy is the lateral and axial resolution. To overcome thislimitation and to achieve higher resolution electron microscopy (EM) apparatus were extensively improved in the lastcentury. In the case of amber, the conventional scanning electron microscopy (SEM) using secondary electron signal(SE) and low temperature scanning electron microscopy (LT-SEM), using either a secondary (SE) or backscattered(BSE) electron detection mode of the signal is furthermore a complementary and very appropriate method [17,18].Since the seventies conventional SEM-SE tools have been applied to study amber organic inclusions mainly duringentomology studies [37]. However there are few reports applying these techniques to the study of includedmicroorganisms [17].

When an electron beam interact with the sample surface different signals are emitted such as low-energy secondaryelectrons, high-energy backscattered electrons, photons and other electromagnetic radiations, as shown in Fig. 4. Thesesignals might be detected by different devices and can be converted for instance into BSE or SE images, and/orelemental maps or spectra, each of which give different characteristics of the sample nature. For SEM-SE observationsthe amber samples are usually coated with gold according to standard procedures [38]. Although the amber maintainedits integrity under the electron beam, the secondary electrons gave only topographical (micromorphology) informationfrom the sample surface (Fig 4a). Prior to SEM-SE examination some amber samples should to be fractured in order toexpose a clean, fresh surface, and then coated with gold. SEM-SE is a useful tool that permits obtain importanttaxonomic information for microorganisms identification, providing also fine structural details of internal tissues andcuticular microsculpture of arthropods included in amber [36,37]. SEM-SE is appropriate for the study of microbiota inamber since the external morphology of microbes can be interpreted as SE signals give contrast images based on thesuperficial topography. An example of SEM-SE visualization is shown in Fig. 5c where a fragment of fungal hyphabecomes to be exposed when amber was fractured.

Moreover, distinction impediment between organic and inorganic phases embedded in resin has been reported [42].Taking into account this impediment of SEM-SE approach in 2003 Ascaso et al. applied for the first time SEM but inbackscattered electrons detection mode (BSE) for visualization of amber inclusions [17]. In this case, a protocol forSEM-BSE sample preparation, previously used for the study of live microorganisms in lithic substrates was adapted[40,41]. In this protocol a polished amber surface or even a polished thin section such as one used for LM was coatedwith carbon and posterior studied with SEM-BSE technique. The behaviour of amber under incident electrons wasoptimal because no damage or other heat alterations were produced after the electron beam had been applied. Thissuccessful approach leads to the interesting results of the entire microcenosis included in amber [17-21]. Thevisualization of microcenosis elements was possible thanks to the mineralization processes undergo by themicroorganisms. In these way even microorganisms lacking a cell wall, such as protozoa, and organisms with cell walls,such as bacteria, fungi and algae embedded in resin, as well as fragments of soft tissues and leaves, could be detected[17-19].

The backscattered electrons signal produce a composite image based on differences in average atomic number (Z) of

the target and derived from a discrete thickness below the sample surface, with an approximate thickness of microns orless [42]. The SEM-BSE images reveal the presence and distribution of different chemical phases and theirultrastructural details that is not evident either in images obtained by light microscopy. The backscatter coefficient of the electrons increases with the increasing of atomic number, thus the SEM-BSE signal shows the difference in Z majorthen 0.01 of the observed structures [43]. Components with higher mean atomic number appear brighter (e.g. fungalhyphae that are mineralised) while structures with a low atomic number (e.g. amber) appear as shade of dark grey (Figs.4b-c, 5b, and 6 e-f). This investigation strategy using SEM-BSE signal is an efficient and successful technique used forthe in situ description and characterization of live and fossilized lithobiontic microorganisms [41,44].

Some of the hyphae when observed in LM were opaque for light and when observed with CLSM do not revealfluorescence and only reflected laser light, Figs. 3d(iii)-f and 6c. These both observations lead to the idea that thesehyphae were mineralized. This suspection was confirmed by SEM-BSE observation where bright (high Z) featurescorresponding to the hyphae structures were localized, Figs. 4 b-c, 5b and 6e-f. This indicates that the hypha core (orlumen) and sometimes the radial sheath have accumulated compounds with a higher atomic number during

mineralization processes. These mineralization processes of organisms included within the Álava amber resin andleading to the formation of iron sulphide compounds was previously reported [21].




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Other type of signals produced by the interaction of the incident electron beam with the specimen are characteristic X-rays. Because carbon is used for coating the samples and this does not interfere with X-ray emission signals, SEM-BSE

Fig. 4 Scheme of the signals detection during SEM visualization and EDS microanalyses after the incident electron beam interactionwith the sample surface. Topographic information and chemical composition of amber sample can be obtained from secondaryelectrons signal, backscattered electrons signal and X-rays using different detectors. a: SEM-SE image of a non mineralized fossilhyphae (arrows) included in the amber matrix. The holes correspond to the lumen zone; b: SEM-BSE image detail of a mineralizedhypha exposing the bright central core and radial filaments; c: results of EDS microanalysis of mineralized fungal hyphae included inamber and visualized by SEM-BSE (left image). The results show qualitative spectrum revelling high amount of Fe and S, andelements distribution maps of Fe and S respectively (centre and right images). All analyzes were performed in Álava amber samplesfrom Salinillas (a) and Peñacerrada outcrops (b and c).

and microanalysis by energy dispersive spectroscopy (EDS) of X-rays could be performed simultaneously, Fig. 4c.Application of EDS microanalysis make possible to obtain qualitative and quantitative composition of mineralizedstructures within the amber resin, see spectra in Fig. 4c. Moreover the elemental map shows the distribution of selectedelements in the observed sample, Fig. 4c. It is necessary to take into account the operating SEM conditions (keyparameters such as accelerating voltage and intensity of electron current) when elemental mapping is carried out (for

further discussion see Orr et al. 2002).As shown in Figure 4c, the qualitative and quantitative analyses of chemical elements indicate that during the fungi

mineralization the deposition of S and Fe was carried in the outside of the hyphal lumen (core) and following thefibrillar structure of the fungal cell wall, formed by chitin and glucan, resulting in the radial sheath shown in 4b.

In our opinion, the SEM-BSE approach in combination with EDS microanalysis demonstrated to be an importantmethod for analysing total or partially mineralized microorganisms and also biological material included in amber [17-21]. This type of analysis provides information about fungi mineralization processes. From a paleontological point of view, this information is very important because it allow us to discern between different modes and degrees of biological preservation and identify the mechanism/s involved. It is necessary to point out that with respect to themicroorganism mineralization/fossilization processes this information is very scarce [17,18,21,45].

5. Low temperature scanning electron microscopy

LT-SEM is a good strategy for exploring microinclusions in amber, especially embedded organisms such as fungi orprotozoa that could contain water [17,18,21]. Moreover this method seems to be highly promising for analysing the

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Fig. 5 Electron microscopy images of fungal hyphae in Álava amber from Salinillas (c, d) and Peñacerrada (a-b) outcrops obtainedby using different ME techniques. a: LT-SEM image of non mineralized fungal hyphae (arrows); b: SEM-BSE image of mineralizedfungal hyphae see the bright core (black arrows) and the radial aspect of the hyphal sheath (white arrows); c: SEM-SE image of nonmineralized fungal hyphae. The holes (arrows) correspond to the lumen zone; d: TEM image of non mineralized fungal hyphae. Thehyphal sheaths can be observed (arrows). Bars: 10 µm.

internal and external appearance of structures that possibly contain water such as liquid-containing inclusions/bubbles.For LT-SEM examination small amber fragments were mounted with O.C.T. compound (Gurr) and mechanically fixedonto the specimen holder using the cryotransfer system (Oxford CT1500). Samples were plunge-frozen in subcooledliquid nitrogen and then transferred to the preparation unit. This is the unique process that can prevent the loss of wateror liquid from the inside of the bubbles. Subsequently, the amber-frozen specimens are fractured in situ and etched at -90 ºC. The exposed surfaces are sputter coated with gold or others elements. Whereas the cryofracture method permits

the observation of the microorganisms trapped in the bubbles or not without contaminants and artefacts whichsometimes are produced by the conventional SEM-SE procedures. The only difficulty using this technique is the verysmall size of the samples to be fractured.

6. Confocal laser scanning microscopy

CLSM offers the possibility of analysing the distribution of microorganisms within the three dimensional space of amber material, and this methodology was applied for the first time to study microcenosis in situ by our group [17-20].Although CLSM detects fluorescent or reflected light exclusively from the focal plane it is possible to obtain imagesthrough the specimen, by sequentially moving the microscope stage in z-direction. The series of all confocal sectionsthat are obtained are calculated using a digital image analysis programme and give a true three dimensional images.Because CLSM techniques allow non-destructive sampling preparation and amber has a generally relatively good

translucency, a three-dimensional (3D) reconstruction of fungal and protozoan has been successfully achieved [17-20].

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Fig. 6 Fungal mycelium in Álava amber samples from Salinillas (a-d), San Just (e) and Peñacerrada (f). a: bright field image of fungal hyphae (white and yellow arrows); b: CLSM image of 3-D reconstructed series of confocal sections of the same zoneobserved by LM in a; note that strong autofluorescence (green, ext/em: 488/515-545 nm) signal proceeded from part of mummified(non mineralized) hyphae (white arrows); c: CLSM image of the same zone observed in a and b but obtained in reflected laser lightrevelling the presence of mineralized hyphae fragments, mainly localized in the hyphae core (yellow arrows); d: CLSM imageproduce by the superposition of b and c images, green colour correspond to autofluorescence hyphae parts and the red colour to themineralized hyphae cores; e: SEM-BSE image of the amber crust showing mineralized fungal hyphae (yellow arrows) and pyritecrystal deposits (white arrow); f : SEM-BSE image of the amber internal zone shows similar to “e” mineralized hyphae (yellowarrows). Bars= a-d: 25µm, f and e: 20 µm.

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In spite of these advantages, CLSM has rarely been use for amber microinclusions analyses [45]. Besides this,autofluorescence signals produced by the non-mineralized biological parts of endogenous organisms included in ambercould be detected [17-21]. It is well known that microorganisms, plants and animals cells and their elements mightcontain organic molecules with fluorescence properties (autofluorescence signal).Resistant biomacromolecules can be preserved in fossil record and some of them can maintain their autofluorescenceproperties even after decay of organisms [47,48]. Chitin is the main structural component of the fungal cell wall

responsible for fungi highly reflective surface, except in the melanized fungi [49,50]. In fungi the autofluorescence isindeed attributed mainly to chitin. Some differences in hyphae autofluorescence has been assigned to chitin changes,such as its type of integration in the cell wall. Depending on the fungal structure chitin is either encased within the outercell wall components or located on the hypha surface [49].

What was awesome surprise it was that we have observed for first time the autofluorescence signal proceeded fromnon mineralized (no SEM-BSE signal) fungal cells embedded within the amber resin. It’s mean that perhaps chitinbearing molecules can still maintain fluorescence properties in non mineralized fungi cells, we have called this structureas mummified cells. These fungal autofluorescence enables excellent CLSM observations and when fungi are includedin amber it can be observed without any manipulation of the specimen as shown in Figure 3d-ii and 6 b. CLSM imagesalso give structural information, e.g. allow the identification of reproductive structures such as sporangia detected infungal mycelium growing on Thysanoptera included in amber and insect amber mold, Fig. 3 dii-ii and e-f.The autofluorescence signal intensity, that can be detected and measured by CLSM, provides information aboutpreservation degree of mummified cells. Those we have indicated the presence of two extreme preservation phases of

fungi cells. On the one side we have found totally mineralized cells visualized by SEM-BSE and analysed by EDStechniques, Fig. 4b-c, 5b and 6e-f. On the other side we have observed fungal cells but without any mineral precipitatesor deposits [17,20,34]. Similar mummified-like cells were highly autofluorescent and perfectly detected by CLSM (Fig.3 dii and 6 b). Many fungal cells show the core mineralized and the part corresponding to the cell walls appeared asmummified and in consequence autofluorescent. In some cases the mineralized core was detected by reflected light of laser and autofluorescent wall cell structure was detected by fluorescence signal in the same visualization procedurewith CLSM, Fig. 6d. The mineralization of fungal core can be confirmed by the study of the same sample by SEM-BSEand EDS. This is the good example of correlative (CLSM-SEM-BSE+EDS) microscopy shown in the Figure 3 d-f and6a-d.

For the CLSM studies polished blocks of amber were used for observation using a LSM 310 Zeiss confocalmicroscope equipped with a Plan-Apochromat 63x/1.40 oil immersion objective. An argon (488 nm) laser was used togenerate an excitation beam and the resultant emission was filtered through Band Pass filter of 515-545 nm. Also aCLSM Leica Microsystem TSP 2 with an argon (488 nm) laser to generate an excitation beam at constant intensity

equipped with a TSP 2 Neofluor oil 20x/0.70 and Plan Apo 63x/ 1.40 oil objectives were used. To obtain 3D images,stacks of 20–30 single confocal optical sections were acquired at 0.5–1 µm intervals through the sample and the imageswere digitally stored and compiled.

7. Transmission electron microscopy

When we began our TEM investigations on the Álava amber, we have difficulty in stabilizing the resin to the electronbeam. Poinar (1992) first addressed this problem while attempting to observe bacteria using TEM of ultrathin sectionsof amber, which underwent severe deterioration. When a thin section is examined using TEM it is susceptible to theheating effect of the electrons and holes can appear. Nevertheless carefully selected TEM conditions (only 60 kVacceleration potential and low current with EM 920 Zeiss TEM) made possible observation of ultrathin sections of theÁlava amber using (unpublished results), see Figure 5d. Note that for purpose of TEM study the ultrathin sections were

prepared only from the amber zone where mummified hyphae were previously observed.Acknowledgements.The authors would like to thank Dr. Luis Alcalá, director of “Fundación Conjunto Paleontológico de Teruel-Dinópolis” for access to specimens and for the support of part of this research, Xavier Delclós (UAB) and Enrique Peñalver (IGME)for providing samples from Teruel outcrops and for the valious information about Palaeobiology and Taphonomy of the SpanishCretaceous amber, and Rafael López del Valle for providing Álava amber samples from MCNA. We also extend our gratitude toFernando Pinto and Teresa Carnota (CCMA-CSIC) for technical assistance, Manuel Castillejo and Jose Manuel Hontoria (MNCN)for some amber sampling preparation, and Alfonso Cortez from (CAI-UCM) for technical assistance. This study has been funded bythe “Diputación Foral de Álava” in the context of several cooperative projects with “Museo de Ciencias Naturales de Álava”, grantsCGL2007-62875-BOS “Ministerio de Educación y Ciencia”, CGL2008-0050 “El ámbar del Cretácico de España: un estudiopluridisciplinar” Ministerio de Ciencia e Innovación, CTM 2009-12838-CO4-03 “Ministerio de Ciencia e Innovación” (Spain) andP631A Project by CSIC. The first author acknowledged a JAEDOC contract of the CSIC (Spain).




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