traditional and new microscopy techniques applied to the...

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. The main advances in this field regarding the study of highly fossiliferous amber deposits of Lower Cretaceous, dated 115-120 Ma old, from Álava and Teruel (Spain) are presented. New approaches using methods as scanning electron microscopy in backscattered electron mode, with energy dispersive X-ray spectroscopy microanalysis, at low temperature and transmission electron microscopy were presented. These techniques give images with exceptional high magnification and resolution 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 novel opportunity for in situ study of amber microorganisms preservation forms and mineralization processes. Fluorescence microscopy has been also successfully applied for detecting fungal autofluorescence in amber. The use of this microscopy techniques have opened the way to study microcenosis included in amber.

Keywords: amber; confocal laser scanning microscopy; fluorescence microscopy; fungi; fossil; light microscopy; low temperature scanning electron microscopy; scanning electron microscopy; scanning electron microscopy in backscattered electron mode; scanning electron microscopy in secondary electron; transmission electron microscopy; x-ray microanalysis.

1. Introduction

First it is necessary to explain the scientific relevance of Spanish amber. This will help us to understand the unique opportunity offered by amber microinclusion in the study of ancient fungi and the obtention of palaeoecological information. 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 fossil bioaccumulation 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 the Iberian 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 and Salinillas (Álava Province), San Just and Arroyo de la Pascueta (Teruel Province) and recently in El Soplao (Cantabria Province) [1, 4]. Early Cretaceous ambers with fossil inclusions are scarce, however such Spanish outcrops are highly fossiliferous consequently which make them of great scientific interest [1]. In general amber is composed by complex mixtures of terpenes that include components that polymerize when exposed to light or oxygen [5]. Some palynological and 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]. Spanish amber varies greatly in form and colour, ranging from yellow, red to dark brown, and from transparent, semitransparent or 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 chemical microenvironments during resin secretion [3]. Fungi is one of the most diverse groups of organisms on Earth, approximately 1.5 million species have been estimated, of which approximately 70.000 have been described [7]. The study of fossil fungi provides valuable information about diversity, structure and evolution of this group of microorganisms [8,9]. These studies have revealed the role play by fungi in the establishment of land plants, in lignin degradation in Devonic forests and the parasitic relationship with plants and animals [10]. However fungal fossils are scarce in geological records because the poor preservation of the fungal structure [11]. Amber is a superb medium for the preservation of fragile organisms even their most delicate structures. This fact explains in part why so many taxa of vertebrates, invertebrates (especially arthropods) and plant inclusions in amber have been reported [12]. However, in spite of the importance of microorganisms in cell evolution and the history of the Earth, fossil resins have not been extensively used as a data source for Paleomicrobiology studies. Studies related to fungi 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 the microscopic 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 amber samples treatment to study microorganisms included in this solid resin [17, 19]. The complexity of amber microinclusion analyses was resolve by using different microscopy strategies. That allowed us to obtain, at the same time, structural, ultrastructural and chemical information about fungi and their preservation processes [17-21]. Here we summarized some of these strategies with the aim of extend in their use in the study of microorganisms included in amber.

Fig. 1 Some examples of Spanish amber. a: Álava yellow amber from Peñacerrada; b: stalactite-shaped, c and d: amber specimens from 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, were investigated 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 in the “Museo de Ciencias Naturales de Álava” (MCNA collection). Amber samples from Arroyo de la Pascueta and Sant Just from Teruel outcrops were also analysed. These samples are housed in the “Fundación Conjunto Paleontológico de Teruel-Dinopólis” (CTP collection). For methodology used in the paleontological excavations for Teruel amber extraction see Delclòs et al. 2007 and Peñalver et al. 2007 [1, 3]. Arthropods are the predominant class of amber fauna found in Spanish outcrops, some fungi associated with insects were also analysed from Álava amber collection [2]. In most cases, amber samples were trimmed and polished manually to minimize light scattering for optimal microscopic observation. The ground and polished amber pieces with inclusions were embedded in an epoxy resin (Epotek 301) to eliminate the ‘‘mirror-effect’’ of internal cracks when illuminated, according to the method previously described by Schlee and Dietrich [23] and used for Cretaceous ambers. This treatment also guarantees the conservation of amber prone 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. The refractive 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 is it 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 fungi included in Spanish amber are also scarce [1, 3, 17-20, 33,34]. Fossilized mycelium and reproductive fungal structures have been observed in Salinillas, Peñacerrada and San Just amber samples using LM, Fig. 2. Some of them are similar to 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 fungal structures 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 (b and d) and La Pascueta (f) outcrops. a: conidiophores with pyriform and smooth phragmoconidio (arrows), two-celled, similar to that present in the actual genus Trichocladium, that includes dematiaceous species of hyphomycetes that occurring on wood in terrestrial or wood submerged in freshwater habitats; b: mature zygospore that resembles of that present in actual zygomycetes; c: fine hyphae with 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 actual genus Geotrichum; e: basidia typically four spored (head arrows) sterigmata (arrow); f: layer of fungal hyphae aggregates form a flat of 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 fungi fluoresce under ultraviolet light and various species could be differentiated by their autofluorescence. The autofluorescence is localized mainly in the cell wall and fungal hyphae septum (see section 6). In our case, FM examination 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 observed by 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 (black arrows) of the same zone observed in b; d: CLSM images of fungal hyphae with terminal sporangium (yellow arrow) localize in amber insect mould; (i), bright field image; (ii) strong autofluorescence (green) signal (ext/em: 488/515-545 nm) proceeded from mummified (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 and parasitism between insects and fungi have played a key role in their evolution [13,35,36]. Some of these relationships between fungi and insects have been observed in amber [11,13-15,25,30,31,35]. In a Thysanoptera specimen included in Álava amber a vegetative mycelium and reproductive fungal structures, similar of that present in actual zygomycetes fungi, 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 this limitation and to achieve higher resolution electron microscopy (EM) apparatus were extensively improved in the last century. 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 during entomology studies [37]. However there are few reports applying these techniques to the study of included microorganisms [17]. When an electron beam interact with the sample surface different signals are emitted such as low-energy secondary electrons, high-energy backscattered electrons, photons and other electromagnetic radiations, as shown in Fig. 4. These signals might be detected by different devices and can be converted for instance into BSE or SE images, and/or elemental maps or spectra, each of which give different characteristics of the sample nature. For SEM-SE observations the amber samples are usually coated with gold according to standard procedures [38]. Although the amber maintained its integrity under the electron beam, the secondary electrons gave only topographical (micromorphology) information from the sample surface (Fig 4a). Prior to SEM-SE examination some amber samples should to be fractured in order to expose a clean, fresh surface, and then coated with gold. SEM-SE is a useful tool that permits obtain important taxonomic information for microorganisms identification, providing also fine structural details of internal tissues and cuticular microsculpture of arthropods included in amber [36,37]. SEM-SE is appropriate for the study of microbiota in amber since the external morphology of microbes can be interpreted as SE signals give contrast images based on the superficial topography. An example of SEM-SE visualization is shown in Fig. 5c where a fragment of fungal hypha becomes 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 in backscattered electrons detection mode (BSE) for visualization of amber inclusions [17]. In this case, a protocol for SEM-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 coated with carbon and posterior studied with SEM-BSE technique. The behaviour of amber under incident electrons was optimal because no damage or other heat alterations were produced after the electron beam had been applied. This successful approach leads to the interesting results of the entire microcenosis included in amber [17-21]. The visualization of microcenosis elements was possible thanks to the mineralization processes undergo by the microorganisms. 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 or less [42]. The SEM-BSE images reveal the presence and distribution of different chemical phases and their ultrastructural 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 major then 0.01 of the observed structures [43]. Components with higher mean atomic number appear brighter (e.g. fungal hyphae 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 for the 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 reveal fluorescence and only reflected laser light, Figs. 3d(iii)-f and 6c. These both observations lead to the idea that these hyphae were mineralized. This suspection was confirmed by SEM-BSE observation where bright (high Z) features corresponding to the hyphae structures were localized, Figs. 4 b-c, 5b and 6e-f. This indicates that the hypha core (or lumen) 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 and leading 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 interaction with the sample surface. Topographic information and chemical composition of amber sample can be obtained from secondary electrons signal, backscattered electrons signal and X-rays using different detectors. a: SEM-SE image of a non mineralized fossil hyphae (arrows) included in the amber matrix. The holes correspond to the lumen zone; b: SEM-BSE image detail of a mineralized hypha exposing the bright central core and radial filaments; c: results of EDS microanalysis of mineralized fungal hyphae included in amber and visualized by SEM-BSE (left image). The results show qualitative spectrum revelling high amount of Fe and S, and elements distribution maps of Fe and S respectively (centre and right images). All analyzes were performed in Álava amber samples from 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 mineralized structures within the amber resin, see spectra in Fig. 4c. Moreover the elemental map shows the distribution of selected elements in the observed sample, Fig. 4c. It is necessary to take into account the operating SEM conditions (key parameters 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 the fibrillar 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 important method 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 the microorganism 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 or protozoa 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 obtained by using different ME techniques. a: LT-SEM image of non mineralized fungal hyphae (arrows); b: SEM-BSE image of mineralized fungal hyphae see the bright core (black arrows) and the radial aspect of the hyphal sheath (white arrows); c: SEM-SE image of non mineralized fungal hyphae. The holes (arrows) correspond to the lumen zone; d: TEM image of non mineralized fungal hyphae. The hyphal 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 fixed onto the specimen holder using the cryotransfer system (Oxford CT1500). Samples were plunge-frozen in subcooled liquid nitrogen and then transferred to the preparation unit. This is the unique process that can prevent the loss of water or 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 which sometimes are produced by the conventional SEM-SE procedures. The only difficulty using this technique is the very small 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 images through the specimen, by sequentially moving the microscope stage in z-direction. The series of all confocal sections that 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 zone observed 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 light revelling the presence of mineralized hyphae fragments, mainly localized in the hyphae core (yellow arrows); d: CLSM image produce by the superposition of b and c images, green colour correspond to autofluorescence hyphae parts and the red colour to the mineralized hyphae cores; e: SEM-BSE image of the amber crust showing mineralized fungal hyphae (yellow arrows) and pyrite crystal deposits (white arrow); f: SEM-BSE image of the amber internal zone shows similar to “e” mineralized hyphae (yellow arrows). 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 amber could be detected [17-21]. It is well known that microorganisms, plants and animals cells and their elements might contain organic molecules with fluorescence properties (autofluorescence signal). Resistant biomacromolecules can be preserved in fossil record and some of them can maintain their autofluorescence properties 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 is indeed 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 outer cell 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 from non mineralized (no SEM-BSE signal) fungal cells embedded within the amber resin. It’s mean that perhaps chitin bearing molecules can still maintain fluorescence properties in non mineralized fungi cells, we have called this structure as mummified cells. These fungal autofluorescence enables excellent CLSM observations and when fungi are included in amber it can be observed without any manipulation of the specimen as shown in Figure 3d-ii and 6 b. CLSM images also give structural information, e.g. allow the identification of reproductive structures such as sporangia detected in fungal 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 about preservation 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 EDS techniques, Fig. 4b-c, 5b and 6e-f. On the other side we have observed fungal cells but without any mineral precipitates or 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 as mummified 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 procedure with CLSM, Fig. 6d. The mineralization of fungal core can be confirmed by the study of the same sample by SEM-BSE and EDS. This is the good example of correlative (CLSM-SEM-BSE+EDS) microscopy shown in the Figure 3 d-f and 6a-d. For the CLSM studies polished blocks of amber were used for observation using a LSM 310 Zeiss confocal microscope equipped with a Plan-Apochromat 63x/1.40 oil immersion objective. An argon (488 nm) laser was used to generate an excitation beam and the resultant emission was filtered through Band Pass filter of 515-545 nm. Also a CLSM 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 images were 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 electron beam. Poinar (1992) first addressed this problem while attempting to observe bacteria using TEM of ultrathin sections of amber, which underwent severe deterioration. When a thin section is examined using TEM it is susceptible to the heating effect of the electrons and holes can appear. Nevertheless carefully selected TEM conditions (only 60 kV acceleration potential and low current with EM 920 Zeiss TEM) made possible observation of ultrathin sections of the Álava 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 Spanish Cretaceous amber, and Rafael López del Valle for providing Álava amber samples from MCNA. We also extend our gratitude to Fernando 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 by the “Diputación Foral de Álava” in the context of several cooperative projects with “Museo de Ciencias Naturales de Álava”, grants CGL2007-62875-BOS “Ministerio de Educación y Ciencia”, CGL2008-0050 “El ámbar del Cretácico de España: un estudio pluridisciplinar” Ministerio de Ciencia e Innovación, CTM 2009-12838-CO4-03 “Ministerio de Ciencia e Innovación” (Spain) and P631A Project by CSIC. The first author acknowledged a JAEDOC contract of the CSIC (Spain).



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