Author response: Dinosaur bonebed amber from an original swamp forest soil

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Dinosaur bonebeds with amber content, yet scarce, offer a superior wealth and quality of data on ancient terrestrial ecosystems. However, the preserved palaeodiversity and/or taphonomic characteristics of these exceptional localities had hitherto limited their palaeobiological potential. Here, we describe the amber from the Lower Cretaceous dinosaur bonebed of Ariño (Teruel, Spain) using a multidisciplinary approach. Amber is found in both a root layer with amber strictly in situ and a litter layer mainly composed of aerial pieces unusually rich in bioinclusions, encompassing 11 insect orders, arachnids, and a few plant and vertebrate remains, including a feather. Additional palaeontological data—charophytes, palynomorphs, ostracods— are provided. Ariño arguably represents the most prolific and palaeobiologically diverse locality in which fossiliferous amber and a dinosaur bonebed have been found in association, and the only one known where the vast majority of the palaeontological assemblage suffered no or low-grade pre-burial transport. This has unlocked unprecedentedly complete and reliable palaeoecological data out of two complementary windows of preservation—the bonebed and the amber—from the same site. Editor's evaluation In an integrative way, the authors introduced an exceptional Konservat-Lagerstätte jointly preserving dinosaur remains and fossiliferous amber. Impressively, this is the first time that strictly in situ amber is reported, and the key claims of the manuscript are well supported by the paleontological and geochemical data. This manuscript will be of broad interest to scientists, including paleontologists, geobiologists, ecologists and geologists, as well as the public. https://doi.org/10.7554/eLife.72477.sa0 Decision letter eLife's review process Introduction Localities preserving either vertebrate bonebeds or fossilised plant resin (amber) are among the most valuable sources of information on past terrestrial ecosystems (Rogers et al., 2007; Seyfullah et al., 2018). Yet, when a bonebed and fossilised resin are found jointly in the same site, and there is certainty that they originally belonged to the same biocoenosis, the potential for extracting and integrating palaeobiological data is barely unmatched in palaeontology. Although amber from the Cretaceous is often found together with other fossils such as plant and, more infrequently, vertebrate remains, fossiliferous amber associated with bonebeds including dinosaurs has been previously reported in only three occasions. Firstly, the lower Cenomanian (ca. 96–100.5 Ma) locality of Fouras/Bois Vert (Charente-Maritime, France) yielded diverse vertebrate remains, including about 50 dinosaur bone fragments, alongside plant macroremains, molluscs, and amber lumps, a few of which were fossiliferous (Néraudeau et al., 2003). From the latter, ~110 bioinclusions belonging to arachnids, springtails and, at least four insect orders have been reported, including several species described (Perrichot et al., 2007; Tihelka et al., 2021). Secondly, amber is known from the upper Campanian (~73 Ma) Pipestone Creek bonebed (Alberta, Canada) (Tanke, 2004; Currie et al., 2008). Although >99% of the 3000 individual fossils recovered from this site belong to Pachyrhinosaurus, other dinosaurs, fish, turtles, lizards, and crocodilians were also found (Currie et al., 2008; Bell and Currie, 2016; Cockx et al., 2020). Six bioinclusions recovered from ca. 50 cm3 of typically <1 cm amber pieces were described (Cockx et al., 2020). Lastly, fossiliferous amber was found in Stratum 11 from the uppermost Maastrichtian (ca. 67–66 Ma) Bone Butte bonebed site (South Dakota, USA) (DePalma, 2010). This site, belonging to the intensively studied Hell Creek Formation, provided ~3000 mostly disarticulated fossils representing >50 species of dinosaurs and other vertebrates; the non-vertebrate material included molluscs, ichnofossils, and plant macroremains, and was mostly found together with the fossiliferous amber (DePalma, 2010; DePalma et al., 2015). The palaeodiversity recovered from the latter, in contrast, was rather scarce, with 22 bioinclusions found in 400 g of collected amber (DePalma, 2010; DePalma et al., 2010; Nel et al., 2010). Other Bone Butte strata yielded non-fossiliferous amber (DePalma, 2010). In addition, a hadrosaur jaw with an amber piece originally attached to it and containing an inclusion was reported from the uppermost Campanian Dinosaur Park Formation in Alberta (McKellar et al., 2019). Further Upper Cretaceous bonebed localities from western Canada yielded amber but lacking bioinclusions (Cockx et al., 2021). The Ariño deposit represents one of the most important Lower Cretaceous dinosaur sites from Europe (Alcalá et al., 2012). This outcrop, located within the Santa María open-pit coal mine (Ariño municipality, Teruel Province, Spain), takes part in the Oliete Sub-basin of the Maestrazgo Basin (eastern Iberian Peninsula) (Salas and Guimerà, 1996). This extensional sub-basin was infilled with sediments deposited in palaeoenvironments ranging from marine to continental during the early Barremian to middle Albian (Meléndez et al., 2000). In this sub-basin, the siliciclastic Escucha Formation, early Albian in age (Peyrot et al., 2007; Bover-Arnal et al., 2016), was deposited overlying Aptian marine carbonates (Cervera et al., 1976). This formation represents coastal environments that included barrier-island systems with back-barrier marshes and flood-tidal deltas (Rodríguez-López et al., 2009). The AR-1 level of the Ariño locality, with ca. 600,000 m2 of surveyed surface, consists of marls with a high concentration of organic matter occasionally forming coal, which underlie the lowest level of coal exploited in the Santa María mine (Figure 1, Video 1; Alcalá et al., 2012). The AR-1 level has yielded a rich and diverse vertebrate fossil record representing more than 10,000 fossils namely found in more than 160 mono- or bitaxic concentrations of usually well-preserved, articulated or semi-articulated partial skeletons (Alcalá et al., 2012; Alcalá et al., 2018; Buscalioni et al., 2013; Villanueva-Amadoz et al., 2015). From these, new species of freshwater and terrestrial turtles, crocodilians, and ornithischian dinosaurs–that is, the ornithopod Proa valdearinnoensis and the nodosaurid Europelta carbonensis– have been described (McDonald et al., 2012; Buscalioni et al., 2013; Kirkland et al., 2013; Pérez-García et al., 2015; Pérez-García et al., 2020). Predatory dinosaurs were also present in the Ariño ecosystem, as evidenced by coprolites, ichnites, and isolated allosauroidean teeth (Alcalá et al., 2012; Alcalá et al., 2018; Vajda et al., 2016). Chondrichthyan and osteichthyan fish remains have also been occasionally found (Alcalá et al., 2012). Regarding the invertebrate record, three ostracod species (Tibert et al., 2013), as well as freshwater bivalves and gastropods, were reported (Alcalá et al., 2012; Kirkland et al., 2013). From the palaeobotanical standpoint, two charophyte species, fern remains, conifer twigs, taxonomically unassigned charcoalified wood remains, undetermined cuticles, and palynomorphs found in both the marls and coprolites (spores, gymnosperm, and angiosperm pollen grains) were previously known (Tibert et al., 2013; Villanueva-Amadoz et al., 2015; Vajda et al., 2016). Based on the former geological and palaeontological data, the Ariño palaeoenvironment was inferred as a freshwater swamp plain with perennial alkaline shallow lakes subjected to salinity fluctuations due to marine influence under a tropical–subtropical climate (Alcalá et al., 2012; Tibert et al., 2013; Villanueva-Amadoz et al., 2015). The level AR-1 was dated as early Albian (ca. 110 Ma) based on charophyte, palynological, and ostracod assemblages (Tibert et al., 2013; Villanueva-Amadoz et al., 2015; Vajda et al., 2016). Figure 1 Download asset Open asset The Lower Cretaceous vertebrate bonebed and amber site of Ariño. (A) Geographical and geological location; modified from Alcalá et al., 2012 (B) Stratigraphic location of the level AR-1; general stratigraphic log from the Oliete Sub-basin, modified from Kirkland et al., 2013, is shown at the left, together with the location of the level AR-1 (red star); a section of the latter, including the stratigraphic location of the amber deposit studied herein, is shown at the right. (C) Santa María open-pit coal mine with indication of the level AR-1 and the two excavated areas rich in aerial amber (yellow dots); the bottom of the open-pit coal is at the right. (D) One of the 160+ bone concentrations found in Ariño, AR-1/10, during vertebrate fieldwork in 2010, showing the holotype of the nodosaurid Europelta carbonensis; metal dustpan ~30 cm long. (E) Root marks at the top of the carbonates below the level AR-1; scale bar, 1 cm. (F) Carbonates right below the level AR-1, displaying edaphic features at the top. (G) Detail photograph of the level AR-1 showing the lower root layer (with amber from resin exuded by roots) and the upper litter layer (with amber from resin exuded by trunk and branches); centimetric scale. See also Video 1. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Amber excavation in the lower Albian bonebed level AR-1 of Ariño during May 2019 and extraction of two strictly in situ (autochthonous) kidney-shaped amber pieces from the root layer. See also Figures 1 and 2. The presence of indeterminate amounts of amber in the AR-1 level from Ariño was first noted by Alcalá et al., 2012, with later works only adding that amber pieces were abundant and sometimes large (Alcalá et al., 2018). The only previously described bioinclusion from Ariño amber was a tuft of three remarkably well-preserved mammalian hair strands corresponding to the oldest hair reported in amber (Álvarez-Parra et al., 2020a). In the Iberian Peninsula, amber is found in Triassic (Ladinian–Rhaetian) and Cretaceous (Albian–Maastrichtian) deposits; those having yielded abundant amber with bioinclusions are mostly late Albian in age, namely from the Basque-Cantabrian (e.g. Peñacerrada I and El Soplao) and Maestrazgo basins (e.g. San Just) (Alonso et al., 2000; Delclòs et al., 2007; Peñalver et al., 2007; Najarro et al., 2009; Peñalver and Delclòs, 2010). Here, we characterise the amber deposit associated with the dinosaur bonebed AR-1 of Ariño from a multidisciplinary standpoint, describing its morphological, geochemical, palaeofaunistic, and taphonomic features, all of which allow us to recognise the palaeontological singularity of this site. Together with complementary palaeontological data (charophytes, palynomorphs, ostracods), our integrative results enable a complete reconstruction of the Ariño biota. Results Amber characteristics Two distinct amber-bearing layers, a lower one and an upper one, are present in the Ariño AR-1 level (Figures 1 and 2, Figure 2—figure supplements 1 and 2). The lower layer overlies a level of carbonates of oligotrophic lacustrine origin showing the development of palaeosols at its top, including root marks (Figure 1E and F). This layer is characterised by abundant, irregular amber lumps (i.e., kidney-shaped) 10–40 cm in length with protrusions, an opaque crust, an inner banding pattern, and lacking bioinclusions (Figure 2A, C and F, Figure 2—figure supplement 1). Aerial amber and charcoalified plant remains are absent in this layer. The kidney-shaped amber pieces are distributed along the exposed area of the AR-1 level and, if not partially exposed due to weathering, are complete. The opaque crust from the amber pieces has an irregular morphology and its ultrastructure shows delicate microprotrusions and no evidence of linear grooves (Figure 2C–E). The banding patterns are formed by variable densities of abundant bubble-like inclusions of different sizes, which are monophasic (solid), biphasic (solid+ liquid), or triphasic (solid+ liquid + gas) (Figure 2G). Mineral crystals have been detected growing inwards within allegedly empty spaces left by larger bubble-like inclusions—these include pyrite cuboctahedrons and needle-shaped crystals from an iron sulphate mineral according to EDS analysis (likely szomolnokite, Fe2+SO4·H2O) (Figure 2H,I, Figure 2—figure supplement 3A). Figure 2 with 3 supplements see all Download asset Open asset Diversity of amber pieces from the AR-1 level and Pleistocene copal pieces for comparison. (A) Kidney-shaped amber piece (root layer). (B) Aerial amber piece (litter layer), corresponding to a resin flow, after partially removing surrounding rock during fieldwork. (C) Kidney-shaped amber piece (AR-1-A-2019.93) from the root layer. (D, E) Two different areas of the external surface from a fragment detached from the piece in (C), showing the preserved delicate surface microprotrusions and no evidence of linear grooves. (F) Kidney-shaped amber piece (root layer) showing the internal banding pattern (AR-1-A-2019.132). (G) Triphasic (solid+ liquid + gas) bubble-like inclusions in a kidney-shaped amber piece (AR-1-A-2019.130). (H) Two pyrite cuboctahedrons in an alleged empty space left by a fluid inclusion (amber piece AR-1-A-2019.86). (I) Needle-shaped crystals from an iron sulphate (likely szomolnokite) growing inward from the walls in an alleged empty space left by a fluid inclusion (amber piece AR-1-A-2019.129). (J) Kidney-shaped piece of Pleistocene copal associated to an Agathis australis root from an overturned stump in Waipapakauri (North Island, New Zealand). (K) Pleistocene copal pieces associated to the root system of the same A. australis stump; coin 2.65 cm in diameter. Scale bars, 2 cm (A–C, F, J), 0.5 mm (D), 1 mm (E), 0.03 mm (G), 0.2 mm (H), and 0.1 mm (I). See also Video 1. The upper layer from the Ariño AR-1 level is rich in amber pieces of flow-, droplet-, and stalactite-shaped morphologies, which often show external and/or internal desiccation surfaces (Figure 2B, Figure 2—figure supplement 2). Small, almost spherical amber pieces about 1–5 cm in diameter, with an opaque crust similar to the kidney-shaped amber pieces, are also present in this layer, yet rare; their surface is polished and more regular in patterning (Figure 2—figure supplement 3B-D). Amber pieces range from translucent to opaque, and from light yellow to dark reddish in colour. One peculiar piece showed subtle, multidirectional surface microscopic scratches and borings, the latter filled with an undetermined material, neither calcium carbonate nor gypsum (Figure 2—figure supplement 3E). The FTIR spectra of two stalactite-shaped amber pieces from Ariño are dominated by a small C-H stretching band at 2925 cm–1, an intense C-H band at 1457 cm–1, and an intense carbonyl band at 1707 cm–1 (Figure 3A, Figure 3—source data 1, Figure 3—source data 2, Figure 3—source data 3), all characteristic of amber (Grimalt et al., 1988). Hydroxyl bands near 3500 cm–1 are present. The Ariño amber spectra are very similar to those from San Just amber, their main difference being the presence of a small band near 1200 cm-1 in the latter. On the other hand, the composition of the organic solvent-extractable materials obtained by GC-MS, comprising the 32.5 % of the Ariño amber, is dominated by labdane resin acids and its diagenetic derivatives, with amberene (I; 1,6-dimethyl-5-isopentyltetralin) being the major component in the bulk extract (Figure 3B, Figure 3—figure supplement 1, Figure 3—source data 4). The labdan-18-oic acids are dominant in the polar fraction of the organic extract from the amber. The identification of the clerodane-family diterpene VI is noteworthy. The analyses show no evidence of significant terpenes of the pimarane/abietane family and discount the presence of ferruginol. The Ariño amber does not show a significant content of either 15-homoamberene (III) or 1-methylamberene (X) (Figure 3B, Figure 3—figure supplement 1; Kawamura et al., 2018). This could point to a lack of the corresponding labdanoid alcohols or non-oxidised C18/C19 labdanoids in the precursor resin, as the diagenesis of these molecules could lead to 1-methylamberene. The decarboxylation of the labdan-18-oic acids prevailing in the Ariño amber polar fraction could be the first step in the diagenesis to amberene and its related compounds, especially isomers of the labdanoid VI, found as a rich distribution of peaks with M+=246. Figure 3 with 1 supplement see all Download asset Open asset Physicochemical characterisation of the Lower Cretaceous amber from Ariño. (A) Infrared (FTIR) spectra obtained from two aerial amber pieces (litter layer); a spectrum from San Just amber (upper Albian) is provided for comparison; arrows indicate the main differences between Ariño and San Just ambers, at around 1200 and 500 cm–1; resolution = 4 cm–1. (B) Gas chromatography-mass spectrometry (GC-MS) trace for the underivatised total solvent extract of aerial amber, showing the structures of the main identified terpenoids, referred herein using Roman numerals (full formulation provided in Figure 3—figure supplement 1B); TMN = trimethylnaphthalenes; the analysed aerial amber is shown at the top right (scale bar 0.5 mm). Figure 3—source data 1 FTIR data of the Ariño amber 1. https://cdn.elifesciences.org/articles/72477/elife-72477-fig3-data1-v1.csv Download elife-72477-fig3-data1-v1.csv Figure 3—source data 2 FTIR data of the Ariño amber 2. https://cdn.elifesciences.org/articles/72477/elife-72477-fig3-data2-v1.csv Download elife-72477-fig3-data2-v1.csv Figure 3—source data 3 FTIR data of the San Just amber. https://cdn.elifesciences.org/articles/72477/elife-72477-fig3-data3-v1.csv Download elife-72477-fig3-data3-v1.csv Figure 3—source data 4 GC-MS data of the Ariño amber. https://cdn.elifesciences.org/articles/72477/elife-72477-fig3-data4-v1.zip Download elife-72477-fig3-data4-v1.zip Bioinclusions A total of 166 bioinclusions were obtained out of 918 g of aerial amber (Figure 4, Figure 4—figure supplement 1, Figure 4—figure supplement 2); about one third of them are well to exceptionally well preserved. Plant inclusions are present, such as numerous fern or conifer trichomes (not considered in the inclusion count) and other undetermined remains (Figure 4—figure supplement 1A-E). The diverse assemblage is chiefly composed of arthropods or remains of their activity, such as spiderweb threads (Figure 4—figure supplement 1F,G) and coprolites, but also a few vertebrate integumentary remains. Arthropods are represented by arachnids and 11 insect orders. Arachnid inclusions belong to mites (Acari) and spiders (Araneae). Mites include a rare trombidiform of the family Rhagidiidae, an oribatid, and an undetermined six-legged larva (Figure 4A). One spider offers taphonomic insights (Figure 4—figure supplement 1H). Five amber pieces with arthropods as syninclusions have spiderwebs preserved; although all are isolated strands, one tangled sample might correspond to a partial web (Figure 4—figure supplement 1F). In the latter, glue droplets on several strands suggest it belonged to an orb web (Figure 4—figure supplement 1G). The insect orders found in the Ariño amber are jumping bristletails (Archaeognatha), crickets (Orthoptera), cockroaches (Blattodea), barklice (Psocodea), thrips (Thysanoptera), whiteflies and aphids (Hemiptera), lacewings (Neuroptera), beetles (Coleoptera), moths (Lepidoptera), gnats, midges, and other flies (Diptera), and wasps (Hymenoptera). Archaeognaths are represented by the inclusion of a cercus and a medial caudal filament. Two orthopterans are poorly preserved, but one could belong to †Elcanidae. A blattodean nymph and an adult have been found, as well as several remains such as probably blattodean isolated antennae. Among the seven psocodeans discovered, new taxa probably within the †Archaeatropidae and Manicapsocidae have been recognised. Thysanopterans are the third most abundant insect order in the Ariño amber, with 11 specimens (Figure 4B and C); three amber pieces contain more than one thrips as One isolated thrips shows a (Figure 4—figure supplement also found in other and an nymph is unusually well preserved (Figure four of and two undetermined of the former have been identified as probably belonging to the (Figure and are preserved in the same amber piece as In addition, an (Figure 4—figure supplement has been found in an amber piece with spiderweb The record consists of two on amber surfaces probably belonging to (Figure 4—figure supplement and a complete which could correspond to a (Figure 4—figure supplement Five specimens have been discovered, two identified as belonging to (Figure 4—figure supplement and (Figure 4—figure supplement A yet is remarkably well preserved (Figure (Figure are represented by specimens of the at least one and probably The first is represented by a well-preserved within the (Figure Lastly, are the most abundant in Ariño amber, for specimens belonging to the and (Figure a new vertebrate inclusion is represented by a with a (Figure Figure 4 with 2 supplements see all Download asset Open asset bioinclusions from the Lower Cretaceous bonebed amber of Ariño. (A) A the oldest known (B) thrips (C) adult thrips (D) A (E) A larva (F) A (G) A the oldest known (H) A preserved (I) A the oldest known (J) A fragment with Scale bars, 0.2 mm (A–C, 0.5 mm (D, F, 1 mm (E), and 0.1 mm and ostracod assemblages from the level AR-1 four species belonging to the and The assemblage is dominated by by well-preserved of (Figure Figure supplement and (Figure Figure supplement for The former is represented by large showing a characteristic and displaying at and such a is variable in other of the same Five of have been based on the at the of the showing the at the by two small and a of (Figure supplement although this had been previously identified in Ariño, of the described herein for the first time in the were (Tibert et al., 2013). several = of belonging to have been recovered (Figure Lastly, rare = of small with to are also present (Figure their remains due to the lack of a et al., 2018). Figure with 1 supplement see all Download asset Open asset and ostracod from level AR-1 of Ariño. and (C) (D) after (E) (F) (G) (H) (I) (J) with indication of and (K) the the at the part of the in right with small (X) of the with left and of the of the with left of the with and left and of the of the with of the with and showing in right in left right Scale bars, 0.5 mm mm 0.2 mm mm and mm See also 1. plant remains are abundant in the upper amber layer (Figure 2—figure supplement correspond to with 2 and Although they are similar to the we to these as due to Other charcoalified wood remains belonging to other have also been a rare sample of plant shows to in and in in diameter, and with walls are but evidence of characteristics suggest that this fossilised might (Figure 4—figure supplement The four studied have provided well-preserved assemblages that include a total of different that is, two from freshwater from of and from pollen and 11 from angiosperm pollen (Figure Figure supplement 1). palynomorphs, of freshwater are a small of the for characterised by the of % of the total (Figure the assemblages for fern such as (Figure (Figure and those of and are namely represented by (Figure related to et al., and the (Figure by and and pollen show amounts for the (Figure which is abundant in The of to is also (Figure (Figure

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