Ancient amino acids from fossil feathers in amber

Ancient protein analysis is a rapidly developing field of research. Proteins ranging in age from the Quaternary to Jurassic are being used to answer questions about phylogeny, evolution, and extinction. However, these analyses are sometimes contentious, and focus primarily on large vertebrates in sedimentary fossilisation environments; there are few studies of protein preservation in fossils in amber. Here we show exceptionally slow racemisation rates during thermal degradation experiments of resin enclosed feathers, relative to previous thermal degradation experiments of ostrich eggshell, coral skeleton, and limpet shell. We also recover amino acids from two specimens of fossil feathers in amber. The amino acid compositions are broadly similar to those of degraded feathers, but concentrations are very low, suggesting that much of the original protein has been degraded and lost. High levels of racemisation in more apolar, slowly racemising amino acids suggest that some of the amino acids were ancient and therefore original. Our findings indicate that the unique fossilisation environment inside amber shows potential for the recovery of ancient amino acids and proteins.

Biomolecules such as proteins have great potential to provide new evidence for investigating ancient fossil organisms 1,2 . Previous studies of ancient proteins have focused primarily on hard tissue remains (e.g. bone and shell) in sedimentary rocks. However, a focus on biomineralised fossils recovered from sediments limits the taxonomic coverage of such analyses [3][4][5][6][7][8][9][10][11] ; large vertebrates are particularly overrepresented. he recovery of proteins from fossils in amber, which are primarily small, terrestrial sot-bodied organisms not found in sediments 12,13 , would provide an important biomolecular archive from extinct taxa that cannot be matched by the sedimentary fossil record.
Fossil inclusions in amber are characterised by exceptional morphological preservation of sot tissues, which suggest the possibility of similarly exceptional protein preservation 13 ; this is supported by two previous investigations of fossils in amber based on levels of amino acid racemisation 14,15 . Amino acids primarily racemise (convert from the let-handed to right-handed chiral forms and vice versa) at N-terminal positions, most racemising very slowly within proteins and relatively more rapidly as free amino acids. herefore the degree of racemisation in a closed system correlates with the degree to which the protein has degraded 3 . Low levels of racemisation as a result of limited peptide bond hydrolysis may imply that peptides are preserved 3 , or indicate some modern protein contamination. Liquid chromatography analysis of insects in resin, copal, and amber samples, ranging in age from 100 years old to 130 million years old, reported ancient amino acids with very low levels of racemisation 14 . Smejkal, et al. 15 14 suggested that amber may provide the ideal environment for protein preservation owing to the dehydrating efects of the resin matrix inhibiting protein hydrolysis.
In this study we provide new data from experimental protein degradation and undertake the irst analyses of racemisation in ancient feathers in amber (Fig. 1). We focus on feathers because they are composed almost entirely of the protein beta-keratin, and the evolution of the various keratin proteins that comprise feathers (and other integumentary structures in vertebrates), have been widely explored and debated 16,17 . hus, identiication of protein sequences from fossil feathers, combined with their morphological investigation, would allow important functional and evolutionary information to be determined over long timescales. Moreover, the preservation potential of keratin in diferent environments has been the subject of many recent papers [18][19][20][21][22][23][24][25][26] . Generally speaking, keratin is thought to have a high preservation potential in sedimentary environments, as suggested by the results of burial experiments and analyses of a number of fossil feathers 18,19,22,[24][25][26][27] . However, experiments focusing on diagenetic alteration report rapid degradation of keratin under increased pressure and temperature 20,23 . Finally, some bacteria are more efective than others at degrading keratin 21 . Based on these apparently contradictory results, the fossilisation of keratin is most likely complex and strongly inluenced by environmental chemistry; investigating the preservation of feathers in the speciic chemical environment in amber will help us further untangle this complex process. In addition to the analysis of fossil feathers, we also conducted thermal degradation experiments on chicken feathers in resin (the precursor to amber) from the modern Araucariacean gymnosperm Wollemia nobilis. his provides crucial additional evidence for interpreting the fossil results by taking into account the environmental framework of protein decay and preservation in resin.

Results and Discussion
Using chiral amino acid analysis by reverse-phase high pressure liquid chromatography (RP-HPLC), we assessed the rate of amino acid degradation during thermal degradation of modern chicken feathers heated dry in modern Wollemia nobilis resin. Despite a slew of recent papers investigating protein fossilisation 3,4,28,29 protein degradation is still not fully understood, and this is especially true in the unusual chemical environment found in amber. he experiments therefore give us a framework to understand the patterns of amino acid racemisation, and changes in amino acid composition, during fossilisation in amber. hese experimentally aged feathers in resin showed very low levels of protein degradation, with D/L < 0.5 for all amino acids in all samples, even ater 504 hours at 140 °C (Fig. 2, Table S3), comparable to or slightly lower than the levels of protein degradation seen in feathers thermally degraded in air (Fig. S1, Tables S4 and S5). Signiicantly, this is considerably slower than rates of protein degradation in comparable experiments on other sample types which contained closed system protein, including ostrich eggshell 28 , coral 29 , and mollusc shells 30 (Fig. 3). he modern chicken feathers in resin also showed a decrease in concentration (Table S2) and a continuous increase in racemisation of amino acids with increased heating time and increased temperature (Fig. 2). www.nature.com/scientificreports www.nature.com/scientificreports/ We also investigated nine fossil specimens (ranging from 44 Ma to 105 Ma in age, Supplemental Table S1, Fig. 1). In seven cases, we could not detect amino acids, suggesting that in these samples all the protein had degraded. Two samples, one each from Eocene (~44 Ma) Baltic amber (Fig. 1A) 31 and Cenomanian (~99 Ma) Burmese amber (Fig. 1E) 32 , had detectable amino acids; of these two samples, we recovered lower concentrations of amino acids from the older Burmese amber feather than from the younger Baltic amber feather (Supplemental Table S2). he primary goal in analysing these amino acids is to rigorously test the null hypothesis that they represent contamination rather than ancient, endogenous, amino acids.
he sampling and analytical methods were carefully developed to prevent introducing modern contamination during our handling of the specimens. he lack of detectable amino acids in seven of our nine samples suggests that this was successful; if modern contamination was introduced during sampling, all samples would likely have been afected.
Although we limited the introduction of contamination during our sampling, modern contamination could also have been introduced by others between the time of collection and analysis. In this case, we would expect the composition of the amino acids to be more similar to a contaminant than to feathers. Human keratin would be the most likely contaminant; previous studies have indicated that contamination of fossils with human keratin primarily occurs through direct skin contact (e.g. the ingerprint/ingertip amino acids) or through keratin dust as part of laboratory dust 33,34 . he proteins present in the ingerprints/ingertips and sweat are primarily human keratin 33,35 ; these both provide a direct test of human keratin contamination. Laboratory dust is a mixture of human keratin and proteins from other sources 33,34 ; including multiple samples of laboratory dust provides a range of comparative mixtures. Other possible contaminants can be introduced during cleaning fossils, keratin from animal hair brushes used to clean fossils or protein-containing glue used to repair fossils; however any brushes would have only made contact with the outer surface of the amber, which we removed prior to analysis, and these samples were not glued. he amino acid composition of the feathers (both fresh and experimentally degraded in resin) does not overlap with any of the contaminant samples including human keratin (Fig. 2), indicating that contamination can be distinguished from feather amino acids. Both fossil amino acid samples are more similar to modern chicken feathers degraded in resin than to dust, ingerprints/ingertips, or sweat, a few of the most likely modern contaminants (Fig. 2), suggesting these amino acids were indeed from the enclosed fossil feather. he Baltic amber sample speciically falls exactly along the line of experimental feather degradation in resin (Fig. 2), indicating that the composition is exactly what we would expect for a feather in resin that is slightly more degraded than in our experiments. he Burmese amber sample does not so clearly match feathers degraded in resin, but it falls between fresh feathers and feathers degraded in resin, and is not similar to any contaminant (Fig. 2).
More evidence that at least some of this signal was from ancient amino acids came from the pattern of amino acid racemisation, which can indicate whether amino acids are modern or ancient. he degree of racemisation in some hydrophobic amino acids, such as Tyr, Val, Phe, Leu, Ile (particularly in the Baltic amber sample), is much higher than would be expected, or indeed than would be possible, for modern contamination (Supplemental   Fig. 2). he fossils have undergone much more extensive decay than the experimental samples, and should therefore have higher D/L values. his is the case for the high D/L values of the hydrophobic amino acids (e.g. Tyr, Val, Phe, Leu, Ile) recovered from the two fossil feathers (Table S3, Fig. 2), which strongly suggests they are (at least in large part) ancient. In contrast, the D/L values of the faster racemising amino acids (e.g. Asx and Ser) are comparatively low, inconsistent with the data from apolar amino acids, and from the experiments; we would typically expect fossils to exhibit high degrees of racemisation of these amino acids, and to preferentially lose polar amino acids as well as the most unstable amino acids 28,30,36,37 . he Burmese amber sample shows less racemisation than the Baltic amber sample (Fig. 2, Table S3), which is unexpected given that Burmese amber (~99 Ma) is approximately twice as old as Baltic amber (~44 Ma). his could be explained by some contamination, or by the degradation of the free amino acids themselves, which would preferentially remove right-handed amino acids from the samples. Signiicant contamination, however, is unlikely due to the strong feather signal in the amino acid composition. Moreover, other researchers have found that keratin proteins preserve even into the Jurassic 27 , suggesting that Cretaceous keratin preservation is not at all unexpected or unreasonable.
he low concentrations of amino acids in all amber samples imply that amber-entombed feathers have undergone signiicant diagenesis. However the variable levels of recovered amino acids further support the idea that not all fossils are equal; that two of nine samples included detectable levels is encouraging. CT and synchrotron images of amber-entombed insects sometimes demonstrate loss of the internal tissue 38 , suggesting that decay proceeds rapidly following entombment when there is still suicient entrapped water to enable decay.
As amino acids from the Burmese amber feather (~99 Ma) are less racemised than the amino acids from the younger Baltic amber sample (~44 Ma), this suggests that the methods of entrapment result in multiple independent diagenetic histories. A number of variables have the potential to afect protein degradation rate. For example, the distinct resin chemistries of diferent ambers are indicative of preservation idelity; some resins are characterised by almost perfect preservation, others by poor preservation 38 . his bias will act on proteins alongside other tissue compositions. In particular, fruit lies decay very quickly in Pinus resin (Pinaceae) and very slowly in Wollemia resin (Araucariaceae) 39 . Baltic and Burmese amber are chemically distinct 31,[40][41][42] . Burmese amber is produced by an Araucareacean, phylogenetically close to Wollemia 42 , and although the botanic source of Baltic amber is disputed 31 , a number of authors have noted it likely belongs to the Pinaceae family 31,43 . It is possible that the decreased racemisation of the older Burmese amber, compared with the Baltic amber, could relect a reduced decay rate due to variations in the amber chemical microenvironment and therefore its antimicrobial efects 44 .
In conclusion, the combination of careful sampling procedures, amino acid composition data, and amino acid racemisation data suggests that we found potentially ancient, endogenous amino acids from fossil feathers in amber. he amino acids we recovered were similar to those previously identiied from insects in Baltic amber 14 : both showed low amino acid concentration, signiicant degrees of racemisation, and the presence of some reactive, polar amino acids. Furthermore, the amino acid composition of the fossils was similar to the amino acid composition of chicken feathers thermally aged in modern resin. However, the D/L values, which were higher in the younger fossil feather than in the older fossil feather, and the high levels of some polar, reactive amino acid in the fossils are not exactly what we would expect based on the experiments. he process of protein fossilisation and degradation in amber requires further study to determine if these discrepancies are due to partial amino acid contamination, or whether the experiments (3 weeks of thermal aging) do not encompass all possible pathways of chemical change and degradation in amber.

Arti cial aging experiments Our artiicial aging experiment on modern feathers used freshly plucked
Gallus gallus domesticus (domestic chicken) feathers which were embedded in freshly extruded Wollemia nobilis resin. Wollemia nobilis trees were purchased from wollemipine.co.uk, and liquid resin was collected from these trees. Wollemia nobilis is an Araucariacean, a family of trees known to produce extensive amber, copal, and resin deposits from the Cretaceous to the Recent, including both Burmese and Spanish amber 40,42,44 ; although there are some chemical similarities between Araucariaceae resin and Baltic amber, the botanic origin of this amber is still widely disputed 30,43,45 . A small amount of liquid resin was dripped onto a glass slide, and chicken feather fragments were then mixed into the resin. he mixture of resin and feather was then coated with more resin, to ensure that the feather was not exposed on the surface of the resin. Heating at 110 °C or 140 °C is commonly used as means of accelerating degradation in order to replicate over laboratory timescales the changes that occur during fossilisation [28][29][30] . Each experiment was thermally aged in a convection oven (Binder) for 1, 2 or 3 weeks at 110 °C or 140 °C. At each sampling interval, feathers in resin were analysed for AAR allowing us to track the rates and patterns of feather protein (keratin) degradation in resin. We did not include environmental water in these experiments, even though that is a common component of such experiments 28-30 , because: (1) in the presence of water, the resin melted at both 110 °C and 140 °C so it no longer protected the feather, and (2) in the previously published closed system experiments mentioned above, the environmental water does not penetrate into the intra-crystalline fraction of amino acids analysed.
Fossil preparation. Fossil feathers in amber (Fig. 1, Supplemental Table S1) were obtained from various sites: two specimens from Cretaceous (Albian) Spanish amber (~105 Ma) 46,47 , CES 426 and CES 457 from the laboratory of the El Soplao Cave, Celis, Cantabria (Spain) encompassing the Institutional Collection from the El Soplao outcrop; six un-numbered specimens from Cretaceous (earliest Cenomanian) Burmese amber (~99 Ma) 32,40 from the Nanjing Institute of Geology and Paleontology in China (numbers were not assigned because they were not holotypes or paratypes); and one specimen from Eocene (Lutetian) Baltic amber (~44 Ma) 31 , SMF Be 370 from Senckenberg Research Institute and Natural History Museum, Frankfurt. Cross contamination was minimised and sampling conducted in an aseptically managed, class 1000 cleanroom at the University of Leicester 48 (typical monitoring of the working environment is better than class 100). All tools that made contact with the amber were SCIENTIFIC REPORTS | (2019) 9:6420 | https doi org s www.nature.com/scientificreports www.nature.com/scientificreports/ cleaned either with a heat treatment (6 hours in a dry furnace at 400 °C) or, for tools that could not survive the heat treatment, a chemical treatment (12% NaOCl (VWR), HPLC-grade water (VWR) and HPLC-grade methanol (VWR)). he surface of each piece of amber, along with any suricial protein contamination, was removed with an aluminum oxide Dremel grinding stone, and then a piece of the amber and feather was removed from the specimen using a synthetic diamond Dremel cutting wheel. Each cut piece was pulverised inside a sterile aluminum foil pocket with a hammer to expose the feather at the surface. Further grinding with an agate mortar and pestle was used to separate the feather from the amber as much as possible, and the fragments of feathers were then transferred to a sterile glass vial for amino acid racemisation (AAR) analysis, to determine if ancient amino acids were present, and if so whether these are still bound into proteins or peptides. An initial, small sample of feather was prepared from each specimen and comprised the target feather and small amounts of amber. hese samples were tested for amino acid yield (see below). Samples that yielded amino acids above the baseline were selected for resampling such that larger pieces of feather were extracted, with care taken to exclude amber from these samples. All amino acid composition results were compared to previously published data on common contaminants including dust 49 , human sweat 50,51 , and human ingerprints/ingertips 33,34,52 . he previously published data on common contaminants included only ten amino acids (rather than the thirteen we analysed) and so, in order to compare our results directly to these data, we recalculated our amino acid composition results using only these ten amino acids.
Amino acid racemisation. Each sample was placed in a sterile glass microvial; 100 µL 7 M HCl (Aristar) was added, the vial was lushed with nitrogen and oven-heated to 110 °C for 24 hours; this is likely to release the maximum concentration of amino acids whilst inducing minimum racemisation 53 . Samples were dried in a centrifugal evaporator. For analysis by reverse-phase HPLC (Agilent 1100) the samples were rehydrated with 50 µL rehydration luid per mg of original sample. he rehydration luid (0.01 M HCl, 1.5 mM sodium azide) contains a non-protein amino acid L-homo-arginine at a concentration of 0.01 mM that elutes with baseline separation approximately 50 minutes into the run time. his was used as an internal standard to quantify the concentrations of amino acids in the sample. Ater rehydration, the samples were spun in a centrifugal evaporator to remove any solid amber debris and the supernatant pipetted of for analysis. he solution was transferred to a sterile autosampler vial with a tapered insert. he amino acid compositions of the samples were analysed by RP-HPLC using luorescence detection following a modiied method of Kaufman and Manley 52 . 2 µL of sample was injected and mixed online with 2.2 µL of derivatising reagent (260 mM N-Iso-L-butyryl L-cysteine (IBLC), 170 mM o-phthaldialdehyde (OPA) in 1 M potassium borate bufer, adjusted to pH 10.4 with potassium hydroxide pellets). he amino acids were separated on a C18 HyperSil BDS column (5 × 250 mm) at 25 °C using a gradient elution of 3 solvents: sodium acetate bufer (solvent A: 23 mM sodium acetate tri-hydrate, 1.5 mM sodium azide, 1.3 µM EDTA, adjusted to pH 6.00 ± 0.01 with 10% acetic acid and sodium hydroxide), methanol (solvent C) and acetonitrile (solvent D). Initially 95% A and 5% C is used at a low rate of 0.56 mL/min, grading to 50% C and 2% D ater 95 minutes. Prior to the injection of the next sample, the column was lushed with 95% C and D for 15 minutes, followed by equilibration of 95% A and 5% C for 5 minutes. he luorescence detector uses a xenon-arc lash lamp at a frequency of 55 Hz, with a 280 nm cut-of ilter and an excitation wavelength of 230 nm and emission wavelength of 445 nm. he L and D isomers of 12 amino acids can be separated, and standards and blanks are routinely analysed. During preparative hydrolysis both asparagine and glutamine undergo rapid irreversible deamination to aspartic acid and glutamic acid respectively 54 . It is therefore not possible to distinguish between the acidic amino acids and their derivatives and they are reported together as Asx and Glx respectively.