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Article

Amber from the Lower Cretaceous of Lugar d’Além Formation, Lusitanian Basin, Western Portugal: Chemical Composition and Botanical Source

by
Thairine Lima dos Santos
1,*,
Mário Miguel Mendes
2,3,4,
Pedro Alexandre Dinis
3,
Pedro Miguel Callapez
4,5,
Pedro Proença e Cunha
3,
Ilunga Tshibango André
6,
Magaly Girão Albuquerque
1 and
Celeste Yara dos Santos Siqueira
1
1
Programa de Pós-Graduação em Química (PGQu), Instituto de Química (IQ-UFRJ), Universidade Federal do Rio de Janeiro, Avenida Athos da Silveira Ramos, Rio de Janeiro 21941-909, Brazil
2
Faculty of Sciences and Technology, Fernando Pessoa University, Praça 9 de Abril, 4249-004 Porto, Portugal
3
MARE—Marine and Environmental Sciences Centre, Department of Earth Sciences, University of Coimbra, Rua Sílvio Lima, 3030-790 Coimbra, Portugal
4
Earth and Space Research Centre—CITEUC, University of Coimbra, Rua do Observatório, 3040-004 Coimbra, Portugal
5
PaleoIbérica Research Group, University of Alcalá, 28805 Alcalá de Henares, Spain
6
Regional Museum of Dundo, Dundo, Lunda Norte Province, Angola
*
Author to whom correspondence should be addressed.
Geosciences 2026, 16(1), 24; https://doi.org/10.3390/geosciences16010024
Submission received: 4 December 2025 / Revised: 26 December 2025 / Accepted: 30 December 2025 / Published: 2 January 2026
(This article belongs to the Special Issue Geosciences, Development and Sustainability: Africa and Europe Together)
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Abstract

The first occurrence of amber from the Lower Cretaceous of the Lusitanian Basin, in the Estremadura region of western Portugal, is here reported. The amber was recovered as isolated clasts in the Portela da Vila outcrop section, near the small villages of Ramalhal and Ameal, from sedimentary deposits belonging to the Lugar d’Além Formation considered to be of Hauterivian age. The chemical composition of amber clasts was examined in order to infer their botanical source via biomarker analysis. GC–MS and GC×GC–TOFMS showed a strong predominance of abietane-type diterpenoids, including compounds such as amberene, ferruginol (phenolic abietane), kaurane and the derivative of clerodane. The dominance of abietane diterpenoids along with these specific biomarkers is consistent with resin exudation by Araucariaceae/Cheirolepidiaceae conifers, as supported by previous chemotaxonomic studies of Cretaceous amber. Palynological studies of the same sedimentary rock samples highlighted a pollen–spore assemblage characterized by low diversity and number of specimens, and dominated by conifer pollen assigned to the genera Classopollis, Araucariacites and coniferous bisaccate pollen, with relative occurrences of fern spores. The combined geochemical and palynological studies strongly support a source related to conifer plants for the amber here reported.

1. Introduction

Amber is a fossilized plant resin produced primarily by coniferous trees, with occasional contributions from other gymnosperms and angiosperms during the Mesozoic and Cenozoic. Its formation begins with resin exudation, often triggered by biotic or abiotic stress, such as insect activity, fungal infection, or mechanical injury [1,2]. Once exuded, the resin undergoes polymerization and oxidation, processes that stabilize it and protect it from microbial degradation. Over geological time, burial under sedimentary layers promotes further chemical transformation through diagenesis, resulting in the hard, chemically altered material recognized as amber [3,4]. The chemical composition of amber, particularly the distribution of terpenoids such as abietanes, kauranes, and labdanes, often retains signatures indicative of its botanical source, allowing paleobotanical reconstruction of the resin-producing flora [5,6,7].
Research conducted over the past few decades has revealed remarkable molecular diversity in fossil resins, with isoprenoid and terpenoid derivatives often appearing as dominant components [4,8,9]. Despite this progress, accurately assigning the botanical affinity of fossil resins remains a significant challenge within chemotaxonomy. This difficulty stems not only from the lack of biomarkers exclusive to certain plant groups, but also from the fact that the extracts analyzed do not always fully reflect the original composition of pre-diagenetic resins, which can limit the representativeness of the results [10]. Thus, identifying compounds with greater discriminatory power remains essential to strengthen the association between chemical signatures and producing taxa.
Among the analytical approaches employed in this context, gas chromatography coupled with mass spectrometry (GC–MS) remains an indispensable tool for detecting molecular markers useful for interpreting the biological origin of resins [6,11,12,13].
The application of GC×GC–TOFMS in the chemical characterization of amber has proven particularly advantageous, as it enables the analysis of intact samples without requiring prior fractionation steps, a procedure still commonly employed in traditional analytical approaches [14]. The two-dimensional separation provides superior chromatographic resolution, allowing the distinction of a wide range of compounds that would typically co-elute in conventional methods [15]. In addition, the high-resolution mass spectrometer integrated into the system helps to discriminate hydrocarbon fragments with very similar masses, enabling the identification of diagnostic ions associated with the chemical structure of the fossil resin. Consequently, the resulting mass spectra act as chemical fingerprints that support the differentiation of resins from distinct botanical sources or varying degrees of alteration [10]. Although direct high-resolution mass spectrometry applied to resins is still an emerging method, expanding studies to include a larger number of samples is expected to strengthen its reliability and enhance its interpretative potential [13,16].
The amber from the Lower Cretaceous of Lugar d’Além Formation was recovered from sedimentary deposits of Portela da Vila site, exposed in the Lusitanian Basin, western Portugal, in a geological context of great relevance for understanding the Iberian Cretaceous and the palaeoenvironmental evolution of the region. The amber that co-occurs with the Early Cretaceous floras of Portugal has not been comprehensively investigated with regard to their chemical composition [17]. Furthermore, this amber constitutes an important archive for stratigraphic and paleobotanical studies, in order to establish correlations with other European amber deposits, expanding our understanding of the distribution and diversity of Mesozoic forests in the Northern Hemisphere.
This study focuses on the molecular characterization of the amber from Portela da Vila, seeking to understand how the set of biomarkers detected may reflect both the botanical source and the palaeoenvironmental setting associated with the formation of this fossilized material.

2. Geological Setting of Sampled Area

The Portela da Vila fossil site, with coordinates 39°07′28″ N; 09°14′51″ W, is situated near the intersection of the N8-2 national road with the A8 motorway from Lisbon to Oporto, approximately 2 km southwest the rural villages of Ramalhal and Ameal, in the municipality of Torres Vedras (west central Portugal) (Figure 1). This area is characterized by a natural landscape in which slightly deformed Cretaceous siliciclastic units extensively crop out. They give rise to a moderate undulating relief, not exceeding 116 m, with alignments of low hills of gentle to moderate slope, drained by a dendritic network of tributaries of the Alcabrichel River.
The local section at Portela da Vila (Figure 2) exposes various Lower Cretaceous non-marine formations representative of the proximal domains of the Lusitanian Basin [18,19,20]. This Mesozoic peri-Atlantic basin of the West Iberian Margin contains a thick and diverse tectono-sedimentary record, in which several rifting phases can be distinguished from the Triassic and Jurassic through the Lower Cretaceous series, with a significant infill of marine carbonates and mixed units interbedded by alluvial sequences with sediments derived from the Variscan Massif of Iberia [21,22,23,24,25].
The above mentioned Lower Cretaceous units of the Lusitanian Basin are widespread across several areas in the southwest of Portuguese Estremadura, between Torres Vedras and Lisbon, where they record a succession of carbonate platform facies interfingered with marginal marine and alluvial domains containing mixed and siliciclastic sediments (Figure 1) [26,27,28]. The corresponding stratigraphic interval is late Berriasian to middle Aptian in age and correlative to the Cretaceous allostratigraphic unit UBS3 defined for the Lusitanian Basin [29,30,31]. The upper boundary of this unit is marked by a major intra-Aptian unconformity, which signals the onset of the transition to a passive-margin context for the West Iberian Margin [32,33,34,35]. The overlying upper Aptian to Campanian–Maastrichtian sequences are organized as a post-rift setting of coarse to fine-grained alluvial siliciclastic units interbedded with the Albian-Turonian West Portuguese Carbonate Platform, formed during a prolonged interval of high sea-levels in the Tethys Realm [36,37,38,39,40,41,42].
According to Rey [41] and subsequent workers, the Lower Cretaceous of the Torres Vedras area is represented by the Torres Vedras Group, comprising six formations, starting with the Vale de Lobos Formation (upper Berriasian–lower Valanginian) and São Lourenço Formation (Valanginian). These units are followed by the Santa Susana Formation (uppermost Valanginian–lower Hauterivian), Lugar d’Além Formation (middle Hauterivian) and Fonte Grada Formation (upper Hauterivian to middle Barremian), all of which are recorded in the Portela da Vila section (Figure 2). The uppermost unit of the area is the Upper Almargem Formation (upper Barremian–middle Albian) [41,42,43,44], not represented locally in the studied exposure.
These siliciclastic formations are dominated by fining-upward sequences of sandstone facies interbedded with pelites, in places rich in plant remains, and represent transitional settings between alluvial or estuarine domains, and the equivalent tidal flat areas of the shallow carbonate platform developed in the Lisbon—Cascais peninsula [26]. In particular, the facies architecture of the Lugar d’Além Formation, which is well represented in Portela da Vila section (Figure 2), indicates a transgressive, high-sea level depositional context ranging from alluvial, deltaic or estuarine environments, to flat areas of nearshore clastic sedimentation proximal to the carbonate platform [43]. These paralic facies, correlative of the Praia dos Coxos Formation of the carbonate platform [28], include levels with brackish invertebrates, such as the Corbulidae bivalves recorded at Portela da Vila.
In contrast, the overlying Fonte Grada Formation records the progradation of coarse alluvial systems represented by gravel and coarse to medium sandstone facies [41]. This unit is exposed in the upper part of the Portela da Vila section, where about 4 m of erosive base alluvial conglomerates and coarse sands with trough cross-bedding overlie the more finely grained facies of the Lugar d’Além Formation.
The samples containing amber examined in this study were collected from lutite beds at the top of the Lugar d’Além Formation, representing the upper part of a finning-upwards sequence of coarse- to medium- and fine, laminated sandstone deposited above marly levels with small Corbulidae bivalves.

3. Materials and Methods

3.1. Sample Collection and Preparation

The amber clasts analyzed here (Figure 3) were extracted from four sedimentary rock samples (Portela da Vila samples 452, 453, 454 and 455) collected by M.M. Mendes and P. Dinis in 2022 in the Portela da Vila site (Figure 1 and Figure 2) close to the small village of Ramalhal, Estremadura region, western Portugal.
The rock samples were first air-dried in the laboratory, disaggregated in water, and washed using a shower through a 125 µm net mesh sieve. The amber clasts and some mesofossil specimens captured on the sieve were cleaned in hydrofluoric (40% HF) and hydrochloric (10% HCl) acids, thoroughly rinsed in water and dried in the air. The amber clasts and plant fossil remains were observed under a NIKON SMZ800 stereomicroscope (Nikon Corporation, Tokyo, Japan).
The palynomorphs were extracted from sedimentary rock samples following standard techniques [44] using concentrated HCl and HF to dissolve carbonates and silicates, and concentrated HNO3 for oxidation. The organic and the mineral material were separated using heavy liquid (ZnCl2). For light microscopy (LM) studies five microscope slides were prepared for each sample. The LM images were taken with a Nikon Coolpix 5400 digital camera (Nikon Corporation, Tokyo, Japan) on a Nikon Eclipse E600 microscope (Nikon Corporation, Tokyo, Japan) using 60× and 100× objectives.
The amber clasts obtained from Portela da Vila site were observed under a Carl Zeiss Discovery V20 stereomicroscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) equipped with an integrated camera. Their characterization considered parameters such as dimensions, morphology, coloration, degree of sphericity, angularity, and translucency. The images captured during the examination are shown in Figure 3.
The amber specimens exhibit irregular shapes, with individual pieces reaching up to 2.8 cm in maximum dimension. Their coloration ranges from brown to orange and yellow, with some specimens displaying darker tones. Both opaque and translucent pieces are present, as well as surfaces that vary from glossy to matte. Sediment inclusions occur within the amber, and some specimens contain small bubbles. No inclusions of small organisms or foliar remains were observed.

3.2. Extraction and Derivatization

Small fragments were subsampled from the original amber specimens to preserve their integrity. These fragments were cleaned with dichloromethane, dried, and pulverized. The pooled powder was extracted by ultrasonication in dichloromethane:methanol (2:1, v/v), centrifuged, and concentrated under nitrogen (N2) to obtain a crude extract. A 10 mg aliquot was derivatized with N-trimethylsilyl-N-methyltrifluoroacetamide (MSTFA) and analyzed by GC–MS and GC×GC–TOFMS.
The preparations and fossil specimens used in this work are housed in the palaeobotanical collections of the Geological Museum of Lisbon, Portugal. The ambers clasts are stored in the University Federal of Rio de Janeiro, Brazil.

3.3. GC–MS Analyses

GC–MS analyses were performed using an Agilent 6890 gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled to a 5973 mass selective detector, equipped with a DB-5 capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness) (Agilent Technologies, J&W Scientific, Santa Clara, CA, USA). Separation was achieved under a programmed oven temperature (60–280 °C at a rate of 3 °C min−1), with helium as the carrier gas and splitless injection. Mass spectra were acquired in full-scan mode under electron impact ionization (70 eV). Compounds were identified by comparison with reference data and the NIST Mass Spectral Database® (v. 2.0, 2005).

3.4. GC×GC–TOFMS

GC×GC–TOFMS was performed using a Pegasus 4D system (LECO Corporation, St. Joseph, MI, USA). Chromatographic separation was achieved with a DB-5MS (30 m × 0.25 mm, 0.25 µm) column (Agilent Technologies, J&W Scientific, Santa Clara, CA, USA) in the first dimension and an HP-17ht (1.0 m × 0.25 mm, 0.15 µm) column (Agilent Technologies, J&W Scientific, Santa Clara, CA, USA) in the second dimension, using helium as the carrier gas (1 mL min−1) and splitless injection. The oven temperature was programmed from 50 to 280 °C at 3 °C min−1, with the secondary oven and modulator operated at higher temperatures than the primary oven. The modulation period was set to 4 s (1 s hot pulse). Data acquisition and processing were carried out using ChromaTOF-GC software (v. 4.51.6).

4. Results

4.1. Palynology

The palynoflora of Portela da Vila is characterized by moderate diversity and generally well-preserved palynomorphs, all representing terrestrial plants (Figure 4).
A total of 1253 spores and pollen grains were recognized in the LM preparations and placed in twenty two genera and 35 species, all representing terrestrial plants.
According to the palynological analysis, the Portela da Vila palynoflora is dominated by conifer pollen (63%) and fern spores (33%). Bryophyte spores accounted 2%, lycophyte spores 2% and no angiosperm pollen grains were recognized in the studied samples.
The most common fern spores are the smooth-walled Cyathidites australis related to the families Cyatheaceae and Dicksoniaceae, and the trilete spores with coarse, compact ridges assigned to Anemiaceae, represented by different species of the genus Cicatricosisporites. Lycophyte spores are not abundant neither diverse. The group is represented by specimens ascribed to the genus Ceratosporites. Among the conifers pollen, the grains attributed to the genera Classopollis (Cheirolepidiaceae), Araucariacites (Araucariaceae) and Callialasporites (Araucariaceae) numerically dominated all the samples. Bissacate pollen grains are also abundant and morphologically diverse in the Portela da Vila palynoflora. Pollen grains attributed to the Cupressaceae (former Taxodiaceae) and Pinaceae are less prominent, represented by the species Spheripollenites psilatus and Cedripites canadensis, respectively.
Noteworthy, the mesofossil flora is characterized by poor preservation of the plant remains and many specimens were too distorted for reliable identification. However, particularly interesting is the abundant presence of vegetative shoots, mainly as coalified compressions and assigned to the species Frenelopsis teixeirae Alvin & Pais and Pseudofrenelopsis Nathorst, both attributed to the extinct conifer family Cheirolepidiaceae (Figure 5).

4.2. GC–MS Analyses

The chromatographic profiles obtained from the GC–MS analysis of amber clasts from Portela da Vila exhibited a diverse range of compounds, some of which were identified based on the mass spectra obtained, while others could not be elucidated. The identified constituents were classified into major chemical groups such as diterpenoids, carboxylic acids, ketones, phenols, and alcohols. Among these, diterpenoids represented the predominant class (Table 1).
The terpenoid composition of the amber is dominated by diterpenoids, particularly abietane and podocarpane derivatives. Among the compounds detected, 16,17,19-trisnorabieta-8,11,13-triene was the most abundant, followed by amberene. A total ion chromatogram of the amber extract with the numbering of the identified compounds is presented in Figure 6. The GC–MS chromatogram of the amber extract shows a light unresolved complex mixture (UCM), appearing as a broad hump beneath the peaks.

4.3. GC×GC–TOFMS

The GC×GC–TOFMS analysis yielded a significantly more resolved and organized chromatographic pattern compared with the conventional GC–MS data. The molecular profile is dominated by terpenoid compounds—primarily diterpenoids, followed by sesquiterpenoids and oxygenated derivatives. Within this distribution, 16,17,19-trisnorabieta-8,11,13-triene remains one of the major constituents, with amberene appearing abundantly as well, in agreement with previous observations.
Beyond these dominant markers, the analysis also revealed homoamberene and several C14 and C15 sesquiterpenoids that were not detected in earlier one-dimensional runs. The two-dimensional separation further enabled the distinction of multiple terpenoid isomers that likely co-eluted under conventional GC–MS conditions. Overall, GC×GC–TOFMS provided a more comprehensive molecular characterization of the Portela da Vila resin, enabling the identification of minor biomarkers and resolving compound classes that remained obscure in one-dimensional analyses (Table S1). The detection of amberene and homoamberene, along with their corresponding mass spectra, is presented in Figure 7A–C.
The chromatographic analyses of the amber revealed a dominant presence of linear alkanes (C13–C28), alongside tricyclic terpenoids, with compounds detected in both native and derivatized forms, indicating incomplete derivatization.
Ferruginol was detected again, although still in low abundance. In addition, derivatized totarol was also identified at low abundance, eluting at approximately 3546 s in the first dimension and 3.4 s in the second dimension.
A clerodane-derived—recognized as a labdanoid biomarker restricted to Araucariaceae among conifers—was detected in the sample, showing a profile comparable to that reported for Dominican amber by [48] (Figure 7D).
Caurane derivatives were also detected (Figure 7E), together with retene and simonellite, indicating the presence of abietane-type diterpenoid derivatives. Together, these molecular markers provide insights into the composition and preservation state of the fossil resin.

5. Discussion

The palynological data from the Portela da Vila deposit indicate a vegetation assemblage dominated by gymnosperms, with a high abundance of pollen attributed mainly to Cheirolepidiaceae and Araucariaceae, accompanied by diverse fern spores. The absence of angiosperm pollen is noteworthy and suggests a floristic setting in which gymnosperms still played a dominant ecological role, consistent with a Mesozoic context. The prevalence of Classopollis points to conifers adapted to stressed conditions, such as coastal or seasonally dry environments, whereas the presence of Araucariaceae indicates warm climatic conditions with sufficient moisture to support forested landscapes. Fern spores further suggest locally humid microenvironments. Overall, the palynological assemblage reflects a heterogeneous paleoenvironment dominated by conifer forests under warm conditions, providing a suitable ecological framework for resin production and preservation.
The diterpenoids originating from labdane and abietane skeletons are widespread among the resins produced by several modern conifer families, which reduces their usefulness as chemotaxonomic indicators [10]. This occurs because their presence reflects a shared chemical trait rather than a specific taxonomic marker or sample contamination. Likewise, α-ionene and methylionene lack diagnostic significance because they result from advanced diagenetic degradation [4,6,49]. Although individual terpenoid biomarkers seldom provide species-level specificity, the overall terpenoid profile—particularly the relative abundance and absence of certain compounds—can yield more meaningful insights into botanical affinity.
16,17,19-trisnorabieta-8,11,13-triene is an aromatic diterpenoid commonly detected in amber, particularly from the Cretaceous period, and serves as a biomarker for the paleobotanical origin. Its occurrence, in combination with the absence of triterpenoid compounds, suggests gymnosperm botanical sources [7,50].
Among the identified compounds, amberene, derived from agathic acid, was detected together with ent-18-norkaurane and labdanes. The co-occurrence of these biomarkers indicates a pattern widely reported for Araucariaceae resins from the Lower Cretaceous [7,9,10,45,51].
The occurrence of ferruginol, in association with dominant non-phenolic labdanoids, suggests that the analyzed resin may derive from Araucariaceae, Cheirolepidiaceae, or Cupressaceae [45,51].
So far, only a few amber occurrences have been tentatively linked to the Cheirolepidiaceae family, and none have been subjected to comprehensive geochemical analysis to confirm a direct association [49]. Developing a chemical signature for resins from the extinct Cheirolepidiaceae is crucial for differentiating between the various resin-producing conifers of the Mesozoic.
In GC–MS analyses, no specific biomarkers characteristic of the botanical families have been identified. This outcome reinforces that GC–MS alone does not provide sufficient resolution to infer botanical sources at the family level, given the compositional overlap and limited specificity of many terpenoid structures. Therefore, higher-resolution and more comprehensive analytical approaches are required to better characterize the chemical profiles of each botanical species and to improve the accuracy of chemotaxonomic interpretations.
The UCM in GC–MS chromatogram of the amber extract indicates the presence of coeluting compounds that the technique cannot fully separate, reflecting the complexity of the resin’s chemical composition.
Although fractionating the sample could potentially improve separation in one-dimensional GC, the loss of extracted material during the fractionation steps is substantial, which would require a larger amount of the valuable sample to perform the analysis.
Method such as comprehensive two-dimensional gas chromatography (GC×GC) can separate coeluting compounds, detect minor biomarkers, and provide a more detailed molecular profile, enabling more accurate chemotaxonomic and palaeoenvironmental interpretations.
The high concentration of terpenoids, partially obscured linear alkanes and alkenes, and also polycyclic aromatic hydrocarbons (PAHs), making their detection by conventional GC–MS difficult; however, GC×GC–TOFMS provided a much clearer separation and identification of these compounds. Overall, GC×GC–TOFMS provides enhanced resolution and sensitivity, enabling detection of low-abundance compounds and offering a more robust framework for assessing botanical affinities in fossil resins.
The overall molecular profile, together with the occurrence of retene and simonellite—both products of aromatization and condensation of abietane-type diterpenoids—strongly supports a conifer source [1,9].
Alkyl-aromatics such as alkyl-benzenes, alkyl-naphthalenes, and alkyl-tetralins represent highly altered diagenetic products derived from a variety of sesquiterpenoid and diterpenoid precursors. Because the original molecular frameworks undergo extensive oxidative modification during diagenesis, many of these compounds can no longer be confidently assigned to specific terpenoid classes [7,47].
The botanical source of amber enriched in amberene and homoamberene, such as the amber studied, has been associated with members of the Araucariaceae, Cupressaceae, and Cheirolepidiaceae families, similar to what has been proposed for French ambers [6,51,52].
The detection of phenolic abietanes in low abundance, such as ferruginol, in the Portela da Vila amber provides an important clue regarding the botanical origin of the resin. This low occurrence contrasts with the two following scenarios: species within Podocarpaceae typically produce high concentrations of phenolic abietane diterpenoids, whereas members of Pinaceae are known to lack these compounds almost entirely [9,10,53,54,55,56]. Therefore, the modest presence of ferruginol and related phenolic abietanes in the analyzed sample suggests that the resin is unlikely to derive from either of these families. Instead, this chemical pattern is consistent with resinous sources from Araucariaceae, Cupressaceae, or Cheirolepidiaceae, all of which can generate abietane diterpenoids in proportions similar to those observed, supporting their plausibility as potential contributors to the studied material [5,9,10,46,55,57,58].
The detection of 15-nor-cleroda-3,12-diene identified in the Portela da Vila amber is highly informative, as such compounds have a narrow taxonomic distribution and are considered strong chemical indicators of Araucariaceae [48]. Its presence therefore provides meaningful evidence supporting an affinity with this family. Additionally, the absence of diagnostic biomarkers associated with other conifer families further reinforces this interpretation and allows for a more confident exclusion of alternative botanical sources for the studied amber. The superior resolution provided by the GC×GC–TOFMS analysis was crucial for revealing this compound and establishing a more robust chemical framework for interpreting the resin’s botanical origin.
A comparative overview of the major Cretaceous amber deposits reported from Spain and France—regions geographically close to Portugal—offers a robust framework for understanding the resin-producing diversity and palaeoenvironmental conditions of western Europe during that time. The Alpine amber of France, dated to the Early Cretaceous, shows affinities with Araucariaceae or Cheirolepidiaceae [45]. In contrast, the extensive Spanish deposits, spanning from the Early to the Late Cretaceous, indicate a mixture of botanical sources, including Cheirolepidiaceae, Cupressaceae, and Araucariaceae [6,51,52,59,60]. Similarly, Charente amber (France), also ranging from the Early to the Late Cretaceous, has been linked primarily to Cupressaceae, although a contribution from Cheirolepidiaceae cannot be ruled out [11,45,61]. Late Cretaceous deposits—such as those from the Pyrenees, Anjou, and the regions of Vendée and Provence—tend to reflect resin signatures dominated by Cheirolepidiaceae, Araucariaceae, or Cupressaceae, depending on the locality [45,62,63]. These records highlight that the western European realm adjacent to Portugal was characterized by the presence of diverse resinous forests providing an essential comparative basis for interpreting Portuguese amber and its potential botanical affinities.
Cedrane- and cuparane-type compounds are considered diagnostic biomarkers of Cupressaceae resins but were not detected in the Portela da Vila amber [9,64]. In contrast to fossil resins from the Czech Republic, whose Cupressaceae affinity was inferred from the presence of cuparene and cedrane, the lack of these biomarkers in the present study points to a distinct botanical source and highlights clear differences in geochemical signatures [65,66].
Amber occurrences in Central Europe reflect a diversity of botanical sources beyond those linked to Araucariaceae and Cheirolepidiaceae. The Late Triassic Lunz amber from Austria has been tentatively attributed to Cupressaceae or Pinaceae [67], whereas the Late Cretaceous ajkaite from Hungary remains of uncertain botanical origin due to limited diagnostic biomarkers [68,69,70]. German deposits show particularly complex origins: the Geiseltal resinites (Middle–Late Eocene) indicate contributions from Cupressaceae (Taxodium) and several resin-producing angiosperms [8,71], while the Bitterfeld amber (Late Oligocene) exhibits a heterogeneous composition derived from multiple gymnosperm and angiosperm families [72,73,74,75].
In addition, according to [16], who also investigated amber using GC×GC–TOFMS, the compound 1,10-dimethyl-2-methylene-trans-decalin was proposed as a chemotaxonomic marker for Cupressaceae; however, this compound was not detected in the Portela da Vila amber.
Diterpenoid compounds such as callitrisic acid and 4-epi-pimaric acid are considered characteristic biomarkers of Cupressaceae, particularly of the genus Callitris, as demonstrated by GC–MS studies showing their consistent presence in resin extracts [11,76,77,78]. In the present study, however, none of these Cupressaceae-diagnostic compounds were detected in the amber analyzed, providing further evidence against a Cupressaceae affinity and reinforcing the interpretation of a distinct botanical origin for the resin.
Valderrama [11] identified four compounds in amber from Archingeay (France) that were interpreted as diagnostic biomarkers suggesting a botanical affinity with either Cupressaceae or Araucariaceae. In contrast, none of these compounds were detected in the Portela da Vila amber. This discrepancy is significant, as the palynological assemblage from Portela da Vila indicates a vegetation dominated by Araucariaceae and Cheirolepidiaceae, with no strong palynological evidence supporting a Cupressaceae contribution. Therefore, the absence of Valderrama’s diagnostic compounds in the Portuguese amber suggests that these molecules may be more closely associated with Cupressaceae resins. Nonetheless, this interpretation must be regarded with caution, because the chemotaxonomic specificity of these compounds remains insufficiently constrained. Broader comparative datasets are still required to determine whether these compounds are truly diagnostic at the family level or reflect regional, ecological, or diagenetic variability.
The integration of palynological evidence with the molecular composition of the amber allows a refined reconstruction of the palaeoenvironment at Portela da Vila. The lack of chemical and palynological indicators of Cupressaceae, together with the stronger affinities to Araucariaceae and possibly Cheirolepidiaceae, suggests that these conifers were the main contributors to resin production within this ecosystem. Overall, the Portela da Vila deposit records a conifer-dominated palaeoforest thriving under warm Mesozoic climatic conditions, with environmental dynamics conducive to amber formation and preservation.
The palaeoenvironmental and palaeoclimatic significance of the Portela amber is broadened when compared with other amber deposits from the Lower Cretaceous of Iberian Peninsula. In several sites of the Iberian Peninsula, the amber formation has been reported in low-energy coastal to deltaic environments along the Aptian–Cenomanian paleoshoreline, under warm and humid climatic conditions [6,59]. These environmental conditions environments supported the presence of mixed forests dominated by conifers assigned to Cheirolepidiaceae, Araucariaceae and Cupressaceae families, ferns and early angiosperms, as documented by palynological and palaeobotanical studies [52,59]. In this context, the Portela da Vila amber data are consistent with a regional palaeoecological model for western Europe, suggesting resiniferous coastal forests adapted to warm climates with fluctuating hydrological conditions, and supporting comparable palaeoclimatic drivers across the western European margin during the Early Cretaceous.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/geosciences16010024/s1, Table S1: GC×GC–TOFMS-Based identification of compounds in the amber clasts.

Author Contributions

Conceptualization, T.L.d.S., M.M.M., P.A.D., P.M.C., P.P.e.C., I.T.A. and C.Y.d.S.S.; methodology, T.L.d.S., M.M.M., P.A.D., P.M.C., P.P.e.C., I.T.A., M.G.A. and C.Y.d.S.S.; validation, T.L.d.S., M.M.M., P.A.D., P.M.C., P.P.e.C., I.T.A., M.G.A. and C.Y.d.S.S.; formal analysis, T.L.d.S., M.M.M., M.G.A. and C.Y.d.S.S.; investigation, T.L.d.S., M.M.M., P.A.D., P.M.C., P.P.e.C., I.T.A., M.G.A. and C.Y.d.S.S.; resources, T.L.d.S., M.M.M. and C.Y.d.S.S.; data curation, T.L.d.S., M.M.M. and C.Y.d.S.S.; writing—original draft, T.L.d.S., M.M.M., P.A.D., P.M.C., P.P.e.C., I.T.A., M.G.A. and C.Y.d.S.S.; visualization, T.L.d.S., M.M.M., P.A.D., P.M.C., P.P.e.C., I.T.A., M.G.A. and C.Y.d.S.S.; Supervision, C.Y.d.S.S.; project administration, T.L.d.S., M.M.M., I.T.A. and C.Y.d.S.S. All authors have read and agreed to the published version of the manuscript.

Funding

This study had the support of national funds through Fundação para a Ciência e Tecnologia, I. P (FCT), under the projects [UIDB/04292/2020] [http://doi.org/10.54499/UIDB/04292/2020] and [UIDP/04292/2020] [http://doi.org/10.54499/UIDP/04292/2020] granted to MARE, [LA/P/0069/2020] granted to the Associate Laboratory ARNET [http://doi.org/10.54499/LA/P/0069/2020].

Data Availability Statement

All data and materials during this study are included in this manuscript and Supplementary Materials.

Acknowledgments

The authors are grateful to Débora de Almeida Azevedo (Instituto de Química—UFRJ) for providing access to the gas chromatography equipment and laboratory facilities at the Federal University of Rio de Janeiro. The authors would like to express their gratitude to the following Brazilian governmental institutions that support scientific research: CAPES, CNPq, and FAPERJ.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:
GC–MSGas Chromatography–Mass Spectrometry
GC×GC–TOFMSComprehensive Two-Dimensional Gas Chromatography–Time-of-Flight Mass Spectrometry
HFHydrofluoric acid
HClHydrochloric acid
HNO3Nitric acid
ZnCl2Zinc chloride
LMLight Microscopy
N2Nitrogen
MSTFAN-methyl-N-trimethylsilyl-trifluoroacetamide
UCMUnresolved Complex Mixture
PAHsPolycyclic aromatic hydrocarbons

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Figure 1. (A). Sketch map of the Iberian Peninsula and the Lusitanian Basin in west central Portugal. (B). Detailed geological map showing the approximate position of the Portela da Vila exposure. The red square indicates the location of the Portela da Vila section.
Figure 1. (A). Sketch map of the Iberian Peninsula and the Lusitanian Basin in west central Portugal. (B). Detailed geological map showing the approximate position of the Portela da Vila exposure. The red square indicates the location of the Portela da Vila section.
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Figure 2. General view of the Portela da Vila site. (A). Stratigraphic log. (B). Panoramic view of the sampled outcrop. Amber were collected from fine-grained beds at the top of Lugar d’Além Formation signed in the log and photo by black arrows. Vertical arrows represent transgressive (black)-regressive (white) trends.
Figure 2. General view of the Portela da Vila site. (A). Stratigraphic log. (B). Panoramic view of the sampled outcrop. Amber were collected from fine-grained beds at the top of Lugar d’Além Formation signed in the log and photo by black arrows. Vertical arrows represent transgressive (black)-regressive (white) trends.
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Figure 3. Amber clasts recovered from the Portela da Vila site, Portugal. Scale bars: 1 mm for all specimens.
Figure 3. Amber clasts recovered from the Portela da Vila site, Portugal. Scale bars: 1 mm for all specimens.
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Figure 4. Transmitted light photomicrographs of some representative palynomophs recovered from the Portela da Vila section. The letters and numbers after each entry are slide references. (A). Cyathidites australis Couper (MS.2 PV 452; sample Portela da Vila 452). (B). Cicatricosisporites hallei Delcourt & Sprumont (MS.4 PV 453; sample Portela da Vila 453). (C). Ceratosporites masculus Norris (MS.3 PV 453; sample Portela da Vila 453). (D). Classopollis noelii (MS.3 PV 454; sample Portela da Vila 454). (E). Cluster of Spheripollenites psilatus Couper (MS.4 PV 453; sample Portela da Vila 453). (F). Araucariacites australis Cookson (MS.2 PV 454; sample Portela da Vila 454). (G). Callialasporites dampieri (Balme) Dev emend. Norris (MS.1 PV 455; sample Portela da Vila 455). (H). Cedripites canadensis Pocock (MS.2 PV 452; sample Portela da Vila 452). (I). Parvisaccites radiatus Couper (MS.2 PV 453; sample Portela da Vila 453). Scale bars: 10 μm for all specimens.
Figure 4. Transmitted light photomicrographs of some representative palynomophs recovered from the Portela da Vila section. The letters and numbers after each entry are slide references. (A). Cyathidites australis Couper (MS.2 PV 452; sample Portela da Vila 452). (B). Cicatricosisporites hallei Delcourt & Sprumont (MS.4 PV 453; sample Portela da Vila 453). (C). Ceratosporites masculus Norris (MS.3 PV 453; sample Portela da Vila 453). (D). Classopollis noelii (MS.3 PV 454; sample Portela da Vila 454). (E). Cluster of Spheripollenites psilatus Couper (MS.4 PV 453; sample Portela da Vila 453). (F). Araucariacites australis Cookson (MS.2 PV 454; sample Portela da Vila 454). (G). Callialasporites dampieri (Balme) Dev emend. Norris (MS.1 PV 455; sample Portela da Vila 455). (H). Cedripites canadensis Pocock (MS.2 PV 452; sample Portela da Vila 452). (I). Parvisaccites radiatus Couper (MS.2 PV 453; sample Portela da Vila 453). Scale bars: 10 μm for all specimens.
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Figure 5. Cheirolepidiaceous remains recovered from the Lower Cretaceous of Portela da Vila section. (A). Vegetative shoot of Frenelopsis teixeirae (specimen P1503; sample Portela da Vila 452). (B). Fragmented shot of Pseudofrenelopsis cf. dinisii (specimen P1507; sample Portela da Vila 455). Scale bar: 500 µm.
Figure 5. Cheirolepidiaceous remains recovered from the Lower Cretaceous of Portela da Vila section. (A). Vegetative shoot of Frenelopsis teixeirae (specimen P1503; sample Portela da Vila 452). (B). Fragmented shot of Pseudofrenelopsis cf. dinisii (specimen P1507; sample Portela da Vila 455). Scale bar: 500 µm.
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Figure 6. GC–MS total ion chromatogram of the Portela da Vila amber. The numbers indicate the identified compounds according to Table 1.
Figure 6. GC–MS total ion chromatogram of the Portela da Vila amber. The numbers indicate the identified compounds according to Table 1.
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Figure 7. (A). Extracted ion chromatogram (m/z 230, 244, and 260) showing the detection of amberene, homoamberene, 15-nor-cleroda-3,12-diene, and ent-18-norkaurane. Mass spectra: (B). amberene. (C). homoamberene. (D). 15-nor-cleroda-3,12-diene. (E). ent-18-norkaurane.
Figure 7. (A). Extracted ion chromatogram (m/z 230, 244, and 260) showing the detection of amberene, homoamberene, 15-nor-cleroda-3,12-diene, and ent-18-norkaurane. Mass spectra: (B). amberene. (C). homoamberene. (D). 15-nor-cleroda-3,12-diene. (E). ent-18-norkaurane.
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Table 1. Chemical composition of the amber specimens based on compound identification by GC–MS.
Table 1. Chemical composition of the amber specimens based on compound identification by GC–MS.
NoClass and Compound NameMFMWRef.
Alkyl benzenes, alkyl naphthalenes, and alkyl tetralins
11,5-DimethylnaphthaleneC12H12156.2I
21,2,3,4-Tetrahydro-1,5,8-trimethylnaphthaleneC13H18174.3I
31-Isopropyl-5-methyl-1,2,3,4-tetrahydronaphthalene C14H20188.3I
45,6,7,8-Tetramethyl-1,2,3,4-tetrahydronaphthaleneC14H20188.3I
52,3,6-TrimethylnaphthaleneC13H14170.3I
6AmbereneC17H26230.4[45,46]
Abietanes and podocarpanes
7Podocarpa-8,11,13-trieneC17H24228.4I
816,17,19-Trisnorabieta-8,11,13-trieneC17H24228.4I
9 16,17,18-Trisnorabieta-8,11,13-trieneC17H24228.4[7,47]
107-Oxo-16,17,19-trisnorabieta-8,11,13-trieneC17H22O242.3[7,47]
11SimonelliteC19H24252.4I
1218-norabieta-8,11,13-trieneC19H28256.4I
13DehydroabietaneC20H30270.5I
1416,17-bisnordehydroabietic acid (a)C18H24O2272.4[47]
Kauranes
15ent-18-NorkauraneC20H34274.5[46]
Phenolic abietans
16FerruginolC20H30O286.5I
Carboxylic acids
17Palmitic acid (a)C16H32O2256.4I
18Oleic acid (a)C18H34O2282.5I
Alcohols and phenols
19MethyltetrahydroionolC14H28O212.4I
204-Butyl-indan-5-olC13H18O190.3I
212-DodecanolC12H26O186.3I
MF: Molecular formula; MW: Molecular weight; I: NIST Mass Spectral Database® (version 2.0, 2005); (a): Analyzed as TMS derivative.
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Santos, T.L.d.; Mendes, M.M.; Dinis, P.A.; Callapez, P.M.; Cunha, P.P.e.; André, I.T.; Albuquerque, M.G.; Siqueira, C.Y.d.S. Amber from the Lower Cretaceous of Lugar d’Além Formation, Lusitanian Basin, Western Portugal: Chemical Composition and Botanical Source. Geosciences 2026, 16, 24. https://doi.org/10.3390/geosciences16010024

AMA Style

Santos TLd, Mendes MM, Dinis PA, Callapez PM, Cunha PPe, André IT, Albuquerque MG, Siqueira CYdS. Amber from the Lower Cretaceous of Lugar d’Além Formation, Lusitanian Basin, Western Portugal: Chemical Composition and Botanical Source. Geosciences. 2026; 16(1):24. https://doi.org/10.3390/geosciences16010024

Chicago/Turabian Style

Santos, Thairine Lima dos, Mário Miguel Mendes, Pedro Alexandre Dinis, Pedro Miguel Callapez, Pedro Proença e Cunha, Ilunga Tshibango André, Magaly Girão Albuquerque, and Celeste Yara dos Santos Siqueira. 2026. "Amber from the Lower Cretaceous of Lugar d’Além Formation, Lusitanian Basin, Western Portugal: Chemical Composition and Botanical Source" Geosciences 16, no. 1: 24. https://doi.org/10.3390/geosciences16010024

APA Style

Santos, T. L. d., Mendes, M. M., Dinis, P. A., Callapez, P. M., Cunha, P. P. e., André, I. T., Albuquerque, M. G., & Siqueira, C. Y. d. S. (2026). Amber from the Lower Cretaceous of Lugar d’Além Formation, Lusitanian Basin, Western Portugal: Chemical Composition and Botanical Source. Geosciences, 16(1), 24. https://doi.org/10.3390/geosciences16010024

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