Taphonomy and Chemotaxonomy of Eocene Amber from Southeastern Australia ⇑ Andrew J

Organic Geochemistry 118 (2018) 103–115

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Organic Geochemistry

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Taphonomy and chemotaxonomy of Eocene amber from southeastern Australia ⇑ Andrew J. Coward a, , Chris Mays a, Antonio F. Patti b, Jeffrey D. Stilwell a, Luke A. O’Dell c, Pedro Viegas a a School of Earth, Atmosphere and Environment, Monash University, Clayton, Victoria 3800, Australia b School of Chemistry, Monash University, Clayton, Victoria 3800, Australia c Institute for Frontier Materials, Deakin University, Locked Bag 20000, Geelong, Victoria 3220, Australia article info abstract

Article history: Amber is a complex, organic polymer that offers unparalleled utility as a preservation medium, providing Received 30 May 2017 insights into past organisms and environments. However, under specific circumstances, this information Received in revised form 28 November 2017 can be compromised through alteration of the amber structure. Understanding the degradation of amber Accepted 10 December 2017 in the geosphere could improve prospecting techniques and maximise the quality and validity of chem- Available online 21 December 2017 ical information from altered samples. This study analysed 114 amber samples retrieved from two new Eocene Australian deposits at Strahan, Tasmania and Anglesea, Victoria using a combination of attenu- Keywords: ated total reflectance Fourier transform infrared and solid-state 13C cross-polarised magic angle spinning Amber nuclear magnetic resonance spectroscopy. Results identified both Class Ib polylabdanoid and Class II Alteration Chemotaxonomy cadinene-based amber. The presence of Class II amber in Australia suggests one of two possibilities: Taphonomy (1) a local Dipterocarpaceae source, the primary producer of Class II resins, despite the absence of this Eocene family from the Australian Eocene fossil record; or (2) a local, unidentified botanical source of Victoria cadinene-based amber. A third alternative, that Class II amber was transported to Australia from Tasmania Southeast Asia via ocean currents, is rejected. Taphonomic analysis revealed four mechanisms of alter- Dipterocarpaceae ation prevalent in amber across the two study regions, with evidence of oxidation and metal carboxylate Fourier transform infrared spectroscopy formation. Both the nature and extent of these alterations were found to vary significantly between Nuclear magnetic resonance spectroscopy classes I and II, suggesting that amber class may play a defining role in determining the chemical pathways by which amber degrades. Of note was the high proportion of amber that exhibited no significant chemical changes despite extensive visible alteration features, suggesting the integrity of palaeobiological and palaeoenvironmental information in these samples may be preserved. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction are controlled by a range of botanical, geological and environmen- tal factors. Botanical source has the largest control of amber com- Amber, also called resinite or fossilised resin, is a polymerised, position, with different groups of conifers and angiosperms organic material originating through the fossilisation of the resins responsible for producing ﬁve distinct resin structural classes of seed plants (Langenheim, 2003). Nearly all plant resins are com- (Fig. 1), as deﬁned by Anderson et al. (1992) and Anderson and posed of different types of terpenoid compounds, complex carbon Botto (1993). The distinct chemical signatures of these structural chains of varying multiples of C5H8 isoprene units; the most com- classes provide a reliable way to determine the botanical sources mon terpenoids found in these resins being mono-, sesqui-, di- and of fossil resins, even long after burial. ‘‘Class I” amber, characteris- triterpenoids (Langenheim, 1969), which consist of two, three, four tic of all extant, resiniferous gymnosperms and select angiosperm and six isoprene units, respectively. In most fossil resins, gradual families, has a macromolecular structure composed of diterpenoid polymerization of diterpenoids or sesquiterpenoids led to the cre- monomers of the labdane skeleton. ‘‘Class II” amber, produced ation of a hardened macromolecular structure, itself encompassing exclusively by angiosperms, is thought to be composed of other occluded, non-polymerised terpenoids. Variations in this ter- sesquiterpenoid monomers based on the cadinene skeleton penoid assemblage, as well as the larger macromolecular structure, (Anderson et al., 1992), although the overall macromolecular structure remains unknown (Anderson and Muntean, 2000). The remaining three classes are rare in the resin fossil record, consist- ⇑ Corresponding author. ing of polystyrene formed from phenolic resins (‘‘Class III”) and E-mail address: [email protected] (A.J. Coward). https://doi.org/10.1016/j.orggeochem.2017.12.004 0146-6380/Ó 2017 Elsevier Ltd. All rights reserved. 104 A.J. Coward et al. / Organic Geochemistry 118 (2018) 103–115

Fig. 1. Skeletal backbones of the five structural classes of amber as defined by Anderson et al. (1992) and Anderson and Botto (1993). various non-polymeric ambers (‘‘classes IV and V”; Anderson et al., degradation pathways. However, some general degradation pro- 1992). cesses have been demonstrated, these being the oxidation of ole- Initial attempts to categorise fossil resins into distinct structural fins into organic acids (Shashoua et al., 2005; Pastorelli et al., classes were undertaken using 13C NMR (Lambert et al., 1985, 2012), acid hydrolysis and saponification of esters (Khanijian 1988, 1990) and pyrolysis–gas chromatography-mass spectrome- et al., 2013; Pastorelli et al., 2013a), depolymerisation of the amber try (Py–GC–MS; Anderson and Winans, 1991; Anderson et al., macromolecular structure (Pastorelli et al., 2013b) and off-gassing 1992). More recently, Fourier transform infrared spectroscopy of formic and acetic acids (Pastorelli, 2011b). These reactions can (FTIR) has also been applied to aid in further subdividing these cat- facilitate changes to the physical characteristics of amber, includ- egories (e.g., Wolfe et al., 2009; Tappert et al., 2011; Seyfullah et al., ing colour, crazing and fragmentation, which may destroy impor- 2015). These recent studies have shown that even very subtle spec- tant palaeobiological data and archaeological artefacts (Bisulca troscopic differences between resin producers can be reliably et al., 2012). The detrimental impact these processes have on detected, and the high number of chemical features these spectra amber in storage remains a major, contemporary problem in the yield demonstrate the utility of these techniques for taxonomic field of amber conservation (Shashoua et al., 2005; Bisulca et al., applications. 2012; Khanijian et al., 2013; Pastorelli et al., 2013b), but the roles These same spectroscopic methods simultaneously yield impor- of these alteration processes on amber within the geosphere have tant clues in discerning the causes and mechanisms of amber alter- remained unexplored. Understanding which factors and reactions ation (Martínez-Richa et al., 2000; Shashoua et al., 2005; Pastorelli, are related to the pre- and post-burial stages of fossil resins, how 2011a). The chemical alteration of amber can be facilitated by a they influence amber degradation and how alteration impacts range of physico-chemical agents, including UV and visible radia- stored biochemical information, may greatly assist in future tion (Williams et al., 1990; Pastorelli et al., 2011; Bisulca et al., prospection and collection of amber with intact palaeobiological 2012;), oxygen (Williams et al., 1990; Shashoua et al., 2005; data. Pastorelli et al., 2012), moisture (Williams et al., 1990; Bisulca This study focused on two newly found, or newly described, et al., 2012; Pastorelli et al., 2013a), high and low pH (Pastorelli Australian localities of Eocene amber. Recently, large quantities et al., 2013a) and common air pollutants (Waddington and Fenn, of amber were recovered from the following sites: (1) the coal 1988; Williams et al., 1990). The highly variable and complex nat- deposits at Anglesea, Victoria; and (2) Strahan, Tasmania; a report ure of amber chemistry renders it difficult to determine exact of fossil resins at the latter site had been made previously by Pole A.J. Coward et al. / Organic Geochemistry 118 (2018) 103–115 105

(1998), but collection and study of these ambers had not yet been 150 m is expressed comprising three stratigraphically discrete coal conducted in depth (more details on these sites in the ‘materials seam groups (C, B and A groups, from lower- to uppermost), inter- and methods’ section below). Both deposits possessed visibly spersed with laterally continuous bodies of fine sandstone and silt- altered amber at varying degrees of degradation despite having stone facies (Holdgate et al., 2001). The sub-environments of the no known prior exposure to the modern atmosphere. The present Eastern View Group have been interpreted by the following four study utilised attenuated total reflectance Fourier transform infra- primary facies associations: (1) meandering river-belt channel- red (ATR-FTIR) spectroscopy and solid-state 13C cross-polarised sandstone facies ( 15 m thick); (2) mouth bar-sandstone facies magic angle spinning nuclear magnetic resonance (13C CP-MAS ( 50 m thick); (3) overbank silt-/claystone facies which constitute NMR) spectroscopy of a large selection of altered and unaltered up to 70% of the unit; and (4) swamp/peat conditions represented ambers from the Anglesea and Strahan deposits. The study has by both isolated lenses and laterally extensive coal seams (Trupp the following aims: (1) to identify the original palaeobotanical et al., 1994). Sandy horizons with shallow fossil roots and overlain sources of amber at Anglesea and Strahan through the determina- by thick coal and carbonaceous mudstone occur near the upper A tion of their structural class; and (2) to establish the chemical Group section at Anglesea coal mine; these are interpreted as pathways by which specific amber classes may degrade in the geo- hydromorphic paleosols (‘histosols’ sensu Mack et al., 1993). At sphere and the potential impact this degradation can have on pre- outcrops of the Anglesea Syncline, including those at Anglesea coal served palaeobiological information. mine, the Eastern View Group is unconformably overlain by chan- nelised sandstone facies of the Boonah Formation (Holdgate et al., 2001). Christophel et al. (1987) identified spore-pollen taxa from 2. Geological setting the A Group coal seam which correlate to the Lower Nothofagidites asperus zone of Stover and Partridge (1973); i.e., Middle Eocene; ca. The amber samples were originally collected from two strati- 49–37 Ma). graphic units of southeastern Australia: (1) the Macquarie Harbour Formation, near Strahan, Tasmania; and (2) the Eastern View Group, Anglesea, Victoria (Fig. 2). 3. Materials and methods The Sorell Basin of western Tasmania, Australia, initially formed during the early phases of extension between the Australian and 3.1. Sample collection Antarctic sectors of Gondwana in the Late Jurassic–Early Creta- ceous (Quilty et al., 2014). The final break-up of these two conti- Amber specimens from the Macquarie Harbour Formation were nents in the Paleogene led to a prolonged phase of regional sampled from five carbonaceous mudstone and lignite deposits subsidence and the deposition of thick sedimentary successions. within the ‘‘unnamed member” described by Pole (1998). These The only onshore component of the Sorell Basin is within the Mac- specimens were collected by J.D.S., P.V., C.M., Mr. W. De Silva and quarie Harbour Graben, strata from which are exposed along the research volunteers from Monash University during field expedi- coastal region of Macquarie Harbour near Strahan, Tasmania tions to the region in February and April, 2014. All collection local- (Baillie, 1989). These exposed sedimentary strata consist of the ities of the Macquarie Harbour Formation were from coastal Macquarie Harbour Formation, overlain by Pliocene–Pleistocene outcrops near Strahan, Tasmania (Fig. 2). Macquarie Harbour For- conglomerate and sandstone (Hill et al., 1997). Correlatives of the mation (near Strahan, Tasmania) field locality codes have prefix Macquarie Harbour Formation have been estimated to be > 1000 ‘‘MH”. m thick from offshore wells (Hill et al., 1997). Segments of the for- All amber specimens of the Eastern View Group were collected mation crop out along the eastern edge of the graben near Strahan, from six horizons within the coal horizons of the ‘‘A Group” coal and preserve two distinct sedimentary packages: (1) the massive seam cropping out at the coal mine at Anglesea, Victoria. These to cross-stratified, quartz sandstone of the lower Strahan Sand sub-deposits have been given the following field codes (prefixes Member; and (2) an overlying, unnamed, fossiliferous member of in parentheses): Unnamed Locality (UL), Block 1 Top (B1T), Block interbedded sandstone and carbonaceous mudstone (Scott, 1960; 1 Lower (B1L), Locality 2 (L2), Locality 3 (L3) and Sump (S). These Pole, 1998). Collectively, these units have a maximum thickness specimens were collected by A.C., J.D.S., P.V., C.M., Ms. A. Pentland of 25 m near the township of Strahan (Pole, 1998), but exceed and research volunteers from Monash University during field expe- 80 m in thickness at coastal outcrops approximately 15–20 km ditions to the region in May and August, 2015. Most of the exam- southeast of Strahan. The facies of the Strahan Sand Member are ined samples were derived from the UL coal horizon, which was typical of fluviodeltaic conditions, but the macro-/microfloral fossil overlying a paleosol characterised by coarse sandstone and shallow content help constrain this interpretation to marginal marine estu- rootlets. aries and mud flats (Pole, 1998); for instance, the region was heav- Processing of bulk sediments and coal collected from both the ily populated by mangroves (Nypa; Pole and Macphail, 1996), and Eastern View Group and Macquarie Harbour Formation uncovered there are intermittent high abundances of marine dinoflagellates in 79 specimens of amber for further characterisation and study. Of these facies (Cookson and Eisenack, 1967; Pole, 1998). Using a these, 53 amber specimens were newly exposed at the initiation combination of pollen (Cookson and Eisenack, 1967; Hill and of the study by manual separation of the specimens from their sili- Bigwood, 1987) and dinoflagellates (Cookson and Eisenack, ciclastic sediment and coal matrices. This ensured the acquisition 1967), outcrops of the Macquarie Harbour Formation near Strahan of fresh samples, unmodified by modern, above-ground processes. have been biostratigraphically correlated to the Malvacipollis diver- The remaining 26 amber specimens had been exposed to indirect sus zone of Stover and Partridge (1973); i.e., Lower Eocene (56–49 sunlight, as well as natural atmospheric temperature and humidity Ma; see summary by Pole, 1998). fluctuations for an indeterminate amount of time prior to the ini- The Eastern View Group is a coal-bearing succession of tiation of the study; once collected, these were stored away from interbedded carbonaceous mudstone and interbedded sandstone natural light. These two groups are subsequently referred to as facies situated within Torquay Basin, Victoria. Onshore, the unit freshly exposed amber and stored amber respectively. Once col- has been estimated at > 500 m within the Anglesea Syncline of lected, each amber specimen and its encompassing sediment the Torquay Basin, and a maximum thickness of > 800 m for off- matrix was inserted into a zip-locked sample bag and stored in a shore successions (Holdgate et al., 2001). The most continuous out- dark, enclosed space at a stable temperature of 26 °C. All samples crop exposure of this unit is at the Anglesea coal mine, where that remain after analysis are housed at Museum Victoria, Aus- 106 A.J. Coward et al. / Organic Geochemistry 118 (2018) 103–115

Fig. 2. (A) Map of Australia; boxed area displayed in Fig. 2B; grey area = emergent. (B) Map of southeastern Australia; grey areas = emergent; boxed areas displayed in Fig. 2C and D. (C) Map of Anglesea coal mine area with amber localities; cross-hatched area = exposed strata; dashed lines = roads. (D) Map of northern Macquarie Harbour region with amber localities; grey areas = emergent; dashed lines = roads. tralia, and provided with museum registration numbers (prefix ity was described according to the transparency of 2 mm thick P252-). fragments. A zone was deemed to be transparent if the movement of a dissecting needle passing directly beneath such a fragment 3.2. Specimen characterisation could be distinguished under a microscope at any magnification. If not, the zone was designated as opaque. Texture was described The physical characteristics of all 79 amber specimens were by observation of individual zones through an optical light micro- examined using a Leica M60 modular stereo microscope, with each scope at a fixed light intensity. Texture categories were assigned amber specimen characterised in terms of smaller sub-divisions per the degree of vitreousness (i.e. the perceived glassiness of the called zones. In this study, a zone is defined as any continuous amber, independent of opacity) and cohesion (i.e. the susceptibility amber section possessing a discrete colour, opacity or texture, with of the amber to powdering or flaking) of each amber zone. Texture multiple zones often co-occurring within a single amber specimen. categories assigned were ‘vitreous’, ‘rough vitreous’, ‘powdery vit- Colour was described through observation of 2 mm thick amber reous’, ‘powdery’, ‘marginal’, ‘dull’, and ‘crazed’. Definitions and fragments, using an optical light microscope at a fixed light inten- representative photographs of each texture are provided in Supple- sity. The colour of each zone was assigned to yellow, orange/ mentary Fig. S2. brown, green, dark brown, or white. Representative examples of Amber specimen zones described in this study were interpreted each colour category are provided in Supplementary Fig. S1. Opac- as altered or unaltered by two complementary methods: (1) A.J. Coward et al. / Organic Geochemistry 118 (2018) 103–115 107 physical alteration, which impacted the observed visual appear- ically ranged from 0.5 to 3 mm3 in volume. Amber was applied to ance of amber (as determined by light microscopy); and (2) chem- the accessory until either the accessory was entirely covered, or ical alteration, which impacted the measured chemistry (as the supply of sample was depleted. Absorbance spectra were measured by ATR-FTIR and/or NMR; see Section 3.4). A zone was obtained across the range of 4000–650 cmÀ1, with 50 scans per described to be physically altered if it did not retain a cohesive, vit- sample at a resolution of 4 cmÀ1. Due to inadequate sample sizes, reous or homogeneous texture. Any zone of physically altered some zones failed to produce bands with an absorption higher than amber was also described to be chemically altered if it: (1) exhib- the chosen limit of quantitation, which was defined as the standard ited substantial, consistent chemical differences from any adjacent deviation of the baseline noise multiplied by a factor of 10. Such zone on the same specimen; or (2) exhibited the same chemical zones were discarded from further analysis. At the conclusion of characteristics to any other amber zone previously classified as analysis for each amber zone, the accessory was then cleaned with chemically altered (as per point #1). Any zone that was neither a drop of acetone and the process repeated for the next zone. physically nor chemically altered was designated as ‘unaltered’. To assess the degree of chemical alteration, the spectra had to be standardised; for this, each spectrum had the baseline at 3.3. Zone selection and preparation 4000 cmÀ1 reduced to zero and the absorbance of the alkyl band at 1378 cmÀ1, which showed only minimal absorbance changes From the 79 amber specimens originally obtained, 114 zones between samples, normalised to one. The band at 1378 cmÀ1 was were selected for chemical analyses by their performance against chosen for calibration purposes over the alkyl band at 1448 two criteria: zone size and homogeneity. For zone size, preference cmÀ1, as used in similar studies (Shashoua et al., 2005; Pastorelli, was given to zones greater than ca. 2 mm in diameter. This was to 2011a), to avoid potential overlap with the broad metal carboxy- ensure there was enough sample to perform all the intended chem- late band at 1570 cmÀ1 evident in some zones. The concentration ical analyses, and, in most cases, reserving adequate material for of a specific bond type was deemed proportional to the relative future replications. For homogeneity, zones which showed perfectly absorbance of the corresponding infrared band, as per Beer’s law. uniform opacity, colour or physical alteration state were preferred, The absorbance of each band was determined through the maxi- as well as those that were absent of all discernible inclusions. mum height of absorption without any baseline corrections, or fur- In preparation for the chemical analyses, each zone was pho- ther manipulations of data. tographed with a Leica M205C stereo microscope equipped with a Leica DFC290 HD digital camera. To isolate each zone, a small 3.4.2. 13C solid-state NMR spectroscopy sample was removed with a fine scalpel. The resulting material, Solid state 13C CP-MAS NMR was conducted on ten amber zones either fine amber powder, or larger, solid fragments, was subse- at the Deakin University Institute for Frontier Materials. To obtain quently observed with an optical light microscope for the presence the spectra, roughly 50 mg of each amber zone was ground to a of any contaminating sediments or other material. If large contam- fine powder and loaded into a 4 mm diameter, zirconia magic inating particles were observed, they were removed with dissect- angle spinning (MAS) rotor. A Bruker Avance III 300 MHz widebore ing tweezers. However, this was impossible to achieve for some spectrometer and 4 mm Bruker HX MAS probe were used, with a powdered amber samples, as the contaminating sediments proved magnetic field strength of 7.05 T corresponding to a Larmor fre- to be too fine and interspersed to remove completely by this quency of 75.5 MHz for 13C. The CP-MAS pulse sequence parame- method. Because of this, control samples of the dominant sediment ters included a 1H90° pulse width of 4 ms and a ramped contact of each collection site (excluding Sump) were prepared, allowing time of 2 ms. A MAS rate of 10 kHz was used with 10,000 scans for the assessment of sediment contamination on the analytical acquired for each spectrum and a 5 s recycle delay. Continuous results by ATR-FTIR. Once isolated from the sedimentary matrix, wave 1H decoupling was also applied during the signal acquisition. the amber material was placed in individual, plastic, sample vials Spectra were processed with 10 Hz of apodisation prior to Fourier and sealed in a UV-resistant desiccator under inert nitrogen gas transformation and phase correction. The 13C chemical shift scale (À0.09 MPa), a relatively constant temperature (19–26 °C) and was referenced to tetramethylsilane at 0 ppm using a secondary removed from ambient light sources. These techniques were reference of solid glycine. adapted from storage recommendations suggested by Pastorelli As in FTIR, each spectrum was standardised. To this end, the res- (2011a) and Pastorelli et al. (2011, 2013b), in order to reduce the onance at 38 ppm in each NMR spectrum was normalised to 1.0. In alteration of any amber samples during the study. Samples were Class I ambers, this resonance has been previously assigned to an returned to the desiccator between each analysis. aliphatic carbon on the bicyclic ring of the labdane skeleton (Fig. 1; Martínez-Richa et al., 2000), and as such is not expected 3.4. Chemical analysis to experience any chemical changes due to alteration.

All 114 amber zones were examined with ATR-FTIR. Of these, 10 zones were also examined using solid state 13C CP-MAS NMR, these 4. Results being the only samples to exceed the minimum required sample mass of 50 mg (the approximate minimum mass for obtaining spec- 4.1. ATR-FTIR tra of acceptable signal-to-noise ratio in < 24 h). The experimental parameters and procedures for each analysis are presented below. Altogether, 114 amber zones were analysed using ATR-FTIR, of which 98 were suitable for further analysis (see Supplementary 3.4.1. ATR-FTIR spectroscopy Table S1 for sample descriptions). 10 spectra were removed due ATR-FTIR spectra were collected at the Monash University to detectable sediment contamination, determined by comparison School of Chemistry using an Agilent Technologies Cary 630 FTIR with the ATR-FTIR spectra of the corresponding sedimentary con- with a diamond ATR accessory. An open-air background spectrum trol. Two additional spectra were removed due to an insufﬁcient of the empty and clean accessory was obtained for each sample. signal to noise ratio for key absorbance bands (see Section 3.4.1). For the spectrum acquisition, a small amount of amber of each A further four spectra were removed due to possessing spectra zone was placed on the accessory and analysed. Due to the small with no relation to either amber or the local sediment. Of these, dimensions of Strahan and Anglesea amber, the quantity of mate- two share a spectrum very similar to that of kaolinite (Bikiaris rial collected from each zone was often low, and sample sizes typ- et al., 2000). 108 A.J. Coward et al. / Organic Geochemistry 118 (2018) 103–115

3400 cm-1 3048 cm-1 1702-1693 cm-1 1640 cm-1 OH stretching CH ethelynic COOH stretching C=C stretching stretching A Re gion e nlar ged Region enlarged in in Figure 3C Figure 3B Type A

Type B Absorbance

Type C

3700 3200 2700 2200 1700 1200 700 Wavenumber (cm-1)

1448, 1378, 1366 cm-1 1320 cm-1 1280 cm-1 1230, 1178 cm-1 1028 cm-1 1007 cm-1 887 cm-1 845 cm-1 793 cm-1 CH2,CH3 bending C-O stretching C-O CH B (esters & acids) stretching ethely nic (alcohols) bending

Type A

Type B Absorbance

Type C

1500 1400 1300 1200 1100 1000 900 800 700

Wavenumber (cm-1)

2951 cm-1 2924 cm-1 2867 cm-1 2847 cm-1 C CH3 CH2 CH3 CH2 stretching stretching stretching stretching

Type A Absorbance Type B

Type C

3050 3000 2950 2900 2850 2800 2750

Wavenumber (cm-1)

Fig. 3. (A) Mean ATR-FTIR spectra of unaltered amber of each spectral type: A (red), B (green) and C (blue). (B) Zoomed in spectra of Fig. 3A from 1500 to 700 cmÀ1. (C) Zoomed in spectra of Fig. 3A from 3050 to 2750 cmÀ1. Notable bands are marked with a line, wavenumber and assignment, if known. Red, green and blue lines represent consistent spectral differences considered especially diagnostic of amber types A, B and C, respectively. Each spectrum has been offset on the y-axis to assist comparison. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) A.J. Coward et al. / Organic Geochemistry 118 (2018) 103–115 109

Table 1 Physical characteristics of amber spectral types A, B and C.

Type No. Sub-deposits Colours (unaltered) Colours (altered) Approx. Amber Alteration Amber Alteration samples size range class patterns textures susceptibility A N = 71 All Anglesea and Yellow, orange/brown, Orange/brown, dark 1–10 mm Class Ib AP 1, 2S All textures Moderate Strahan deposits white and dark brown brown, white, yellow and 2A B N = 18 UL and B1L at Green. Appears black in Dark brown, white 5–25 mm Class II AP 3 and 4 Vitreous and Low Anglesea. large samples powdery C N = 9 L2 at Anglesea. Orange/brown Yellow 10–30 Unknown AP 1 Vitreous, dull Very high mm and powdery

No. samples represents the total number of zones to undergo successful chemical analysis. Amber susceptibility represents proportion of total type population to exhibit alteration textures. Images of colour and texture categories provided in Supplementary Figs. S1 and S2.

4.1.1. ATR-FTIR: Amber chemotaxonomic groups classified as either ‘powdery’ or ‘powdery vitreous’. Alteration pat- ATR-FTIR analyses identified three distinct populations of Stra- tern 2S was found in five Type A zones from Strahan, and was char- han and Anglesea amber based on substantial differences between acterised primarily by increases in the broad hydroxyl band at their spectra (Fig. 3). Each population set corresponds directly to 3400 cmÀ1, increased absorbance from 1630 to 1500 cmÀ1, possi- particular colour and size distributions (Table 1), but remain inde- bly due to the C–O asymmetric stretching of metal carboxylates pendent of the amber alteration state. Based on subsequent NMR (Khanijian et al., 2013), and decreased absorbance in the C–O band analysis, it is hypothesised that each of these groupings represents of esters and acids at 1230 and 1178 cmÀ1, in the carboxyl band at amber derived from a distinct botanical source (see Section 5.1). 1693 cmÀ1 and in the alkyl stretching band from 3000 to 2800 For the purpose of discussion, each group will be designated Type cmÀ1. Alteration pattern 2A, in addition to the same absorbance A, Type B and Type C in order of decreasing group size. changes characterising alteration pattern 2S, was defined by an All amber types were found to share the key absorption features additional, substantial drop in exomethylene absorbance at 887 commonly indicative of amber (Fig. 3; Guiliano et al., 2007; Dutta cmÀ1. It was identified only at Anglesea in seven Type A zones. et al., 2009; Tappert et al., 2011). Differences between amber types Excluding one yellow zone, pattern 2S and 2A zones were exclu- were instead expressed as large changes in the relative absorbance sively orange/brown and dark brown in colour. Both patterns col- of major bands, as well as the presence or absence of numerous, lectively possessed a higher cohesion relative to pattern 1, with minor absorbance bands (coloured lines, Fig. 3). Type A amber just three of 12 zone described as either ‘powdery’ or ‘powdery vit- was characterised by four additional bands at 2847, 1320, 845 reous’. Alteration pattern 3 remained exclusive to ten Type B and 793 cmÀ1, none of which were present in Type B or C amber. amber zones found at Anglesea and was defined by increased Type B amber was characterised by unique C-H stretching and absorption of the carboxyl band at 1702 cmÀ1 and of the C–O bending bands at 2951 and 1366 cmÀ1 respectively, in addition stretching of esters and carboxylic acids at 1245 and 1170 cmÀ1, to abnormally low intensity carboxyl and C–O stretching bands in addition to slight decreases in exomethylene absorption at at 1700, 1230 and 1178 cmÀ1, which, unlike types A and C, were 880 cmÀ1 and alcohol and ether absorption at 1028 cmÀ1. Visually, never observed to exceed the absorbance of the adjacent band at pattern 3 was expressed entirely through colour change; in a given 1028 cmÀ1 in any spectrum. Type C amber was defined by addi- amber specimen this was expressed as a concentric ring of dark tional bands at 1280 cmÀ1 and 1007 cmÀ1, in addition to being brown, vitreous amber encompassing an unaltered core of green, the only amber type to exhibit a higher absorbance of the 1378 vitreous amber. Alteration pattern 4 was found on the exterior of cmÀ1 band relative to the adjacent 1448 cmÀ1 band. two Type B specimens as a white zone of ‘powdery’ texture. This pattern exhibited very unusual absorption changes relative to the unaltered portion of the resin; including the appearance of broad 4.1.2. ATR-FTIR: Amber taphonomy and alteration patterns metal carboxylate absorption at 1500–1650 cmÀ1, increased The ATR-FTIR spectra of physically altered amber zones from absorption of the alkyl bending at 1448 cmÀ1, the shifting of the Anglesea and Strahan were directly compared to the averaged alkyl stretching band at 2908–2928 cmÀ1 and the appearance of absorbance of all unaltered amber zones sourced from the same several unidentified absorption bands between 820 and 765 cmÀ1. deposit and amber type. Using the ATR-FTIR spectra of all 45 unaltered amber zones, preliminary comparisons of vitreous opaque vs 4.2. 13C NMR transparent amber, and freshly exposed vs stored amber found no significant spectral differences between zones. As such, these vari- Ten 13C NMR spectra were obtained, including representative ables were not treated as separate datasets. spectra from each of the three amber types (A, B and C; Fig. 5) By comparison against unaltered zones, Strahan and Anglesea and from four of the five alteration patterns (1, 2A, 3 and 4; ambers were found to exhibit four main categories of chemical Fig. 6) identified previously by ATR-FTIR. No spectrum representa- alteration, as defined by their ATR-FTIR spectra, with each category tive of alteration pattern 2S was obtained, as no zone in this pat- experiencing either none, small or substantial levels of absorbance tern possessed enough sample to meet the minimum sample changes in key bond types. The categories were designated alter- mass requirements for NMR. 13C NMR was employed to test the ation patterns (AP) 1–4, with alteration pattern 2 further subdi- validity of the chemotaxonomic and taphonomic categories identi- vided into two subclasses, 2A and 2S (Fig. 4). fied by ATR-FTIR spectra and optical properties; however, due to Alteration pattern 1 was detected in twelve Type A zones from the small number of samples employed herein, the 13C NMR results Strahan, and in two Type A and all seven altered Type C zones from should be considered exploratory rather than definitive. Refer to Anglesea. It is defined as having no significant absorbance changes Supplementary Table S1 for further sample descriptions. between the mean ATR-FTIR spectra of altered and unaltered amber, despite extensive physical alteration. Visually, pattern 1 4.2.1. NMR: Amber chemotaxonomic groups zones were predominately yellow, orange/brown and white in col- Three distinct spectral types, labelled A, B and C as previously our. In terms of alteration textures, pattern 1 exhibited low cohe- defined by ATR-FTIR, were also evident in the 13C NMR spectra sion relative to other patterns, with 10 of 14 Type A textures (Fig. 5). All but one spectrum (855; representative of alteration 110 A.J. Coward et al. / Organic Geochemistry 118 (2018) 103–115

Alteration Pattern 1 (Type A amber)

Alteration Pattern 1 (Type C amber)

Alteration Pattern 2S

Alteration Pattern 2A Relative absorbance

Alteration Pattern 3

Alteration Pattern 4

3700 3200 2700 2200 1700 1200 700

Wavenumber (cm-1)

Fig. 4. Mean ATR-FTIR spectra of alteration patterns 1 (types A and C), 2S, 2A, 3 and 4, each plotted (black) relative to the mean ATR-FTIR spectrum of all unaltered amber zones of an equivalent type and deposit (grey; from top, Type A Strahan, Type C Anglesea, Type A Strahan, Type A Anglesea, Type B Anglesea, Type B Anglesea). For comparison purposes, all spectra have been offset on the y-axis, had the baseline at 4000 cmÀ1 reduced to zero and the absorbance of the peak at 1378 cmÀ1 normalised to 1.0. pattern 4) exhibited a distribution of peaks analogous to previous 4.2.2. NMR: Amber taphonomy and alteration patterns NMR studies (Grimalt et al., 1988; Martínez-Richa et al., 2000; Amber zones previously characterised by ATR-FTIR as alteration Lambert et al., 2005). In general, the aliphatic region extended pattern 1 displayed no significant spectral differences relative to from 10 to 62 ppm, comprising six to 10 peaks corresponding the NMR spectra of unaltered zones of a comparable type (Fig. 6), to methyl and methylene carbons and, from 46 to 62 ppm, car- showing good consistency with ATR-FTIR results. The zone previ- bons bonded to primary and secondary OH groups. Tertiary alco- ously characterised by ATR-FTIR as alteration pattern 3 likewise hols are further evident at 86 ppm in the Type C spectrum. The showed no significant spectral differences in NMR relative to unal- olefinic region comprised four peaks extending from 100 to 150 tered fragments from an adjacent zone, despite large differences in ppm; unsubstituted and disubstituted exomethylene carbons at carboxyl concentration between the same zones in FTIR. This is due 106 ppm and 147 ppm, respectively, absent or extremely weak to the absence of any carboxyl peak in all Type B NMR spectra, in Type B spectra, and two endocyclic carbons at 125 ppm and likely caused by the lack of any adjacent hydrogens to the carboxyl 134 ppm. The carbonyl region ranged primarily from 170 to carbon. The zone characterised previously as alteration pattern 2A 190 ppm in Type A and C spectra, with an additional, weak peak showed reduction in the intensity of the carbonyl and exomethy- at 210 ppm in the Type C spectrum, thought to correspond to the lene resonances, as well as increases to primary and secondary carbons of aldehyde and ketone groups. The most prominent peak alcohol resonances at 52 and 49 ppm, relative to unaltered frag- in this region, at 184 ppm, represented the carbonyl carbon of ments from an adjacent zone. The zone characterised previously carboxylic acids, although this peak was significantly offset in as alteration pattern 4 exhibited a very unusual spectrum, as in the Type C spectrum. No peaks were present within the carbonyl ATR-FTIR, consisting of a range of sharp, well-resolved peaks in region of any Type B spectrum. the aliphatic and olefinic regions. The extensive spectral A.J. Coward et al. / Organic Geochemistry 118 (2018) 103–115 111

Type A

Type B

Type C

200 150 100 50 0

ppm

Fig. 5. 13C NMR spectra of three representative amber zones corresponding to the three spectral types A, B and C as previously identiﬁed by ATR-FTIR. Each spectrum has been offset on the y-axis to assist comparison.

Alteration Pattern 1 (Type A amber)

Alteration Pattern 2A

Alteration Pattern 3

Alteration Pattern 4

220 200 180 160 140 120 100 80 60 40 20 0

ppm

Fig. 6. 13C NMR spectra of four altered amber zones (black), with representative zones from alteration patterns 1, 2A, 3 and 4 as previously identiﬁed by ATR-FTIR. Each pattern has been plotted relative to a representative 13C NMR spectra of an unaltered zone from the same amber type and deposit (grey). For comparison purposes, all spectra have been offset on the y-axis and the resonance at 38 ppm normalised to 1.0. Note for AP 2A and 3, both altered and unaltered zones were obtained from the same amber specimen.

differences between this spectrum and that of unaltered Type B 5.1.1. Type A amber prevent any clear quantitation of chemical change. The NMR spectrum for Type A amber, recovered from all localities in both Anglesea and Strahan, corresponds closely with Group A amber as described by Lambert et al. (2015), sharing a character- 5. Discussion istically similar aliphatic band differing only in the especially high intensity of the 43 ppm peak. The olefinic and carbonyl regions 5.1. Chemotaxonomy of amber at Anglesea and Strahan likewise also correspond closely. The assignment to Group A, which corresponds to Class Ib amber, aligns with other South Paci- Both ATR-FTIR and 13C NMR analysis of Strahan and Anglesea fic ambers which have also been placed into the group, including ambers identified three discrete types of spectra, each likely corre- New Zealand ambers previously associated with Agathis australis sponding to a distinct botanical source. Through comparison of the (Lambert et al., 1993; Lambert and Poinar, 2002; Lyons et al., NMR spectrum of each amber type to an existing classification pat- 2009; Seyfullah et al., 2015) and Australian ambers recovered from tern by Lambert et al. (2015), the structural class of that sample Latrobe Valley, Victoria (Lambert et al., 1993; Lyons et al., 2009). can be identified. This result can then be generalised to other sam- Specifically, the 13C NMR spectra of Type A amber bears an espe- ples in the study, based on similarities between ATR-FTIR spectra. cially close resemblance to the normal decoupled spectrum of Lambert et al. (2015) classified amber into five groups on the basis the resin of the extant Agathis lanceolata (Araucariaceae), as pre- of differences between NMR spectra. These groups were labelled A sented by Lambert and Poinar (2002). Likewise, the ATR-FTIR spec- through E, and when correlated to the amber structural groups of tra of Type A amber was also comparable to possible Agathis Anderson et al. (1992), defined by Py-GC–MS, corresponded to australis amber from New Zealand (Lyons et al., 2009), with all À classes Ib, II, Ia, Ic and III respectively (Lambert et al., 2008). major absorption peaks above 887 cm 1 shared between all 112 A.J. Coward et al. / Organic Geochemistry 118 (2018) 103–115 spectra. However, this evidence does not discount a rarer Cupres- with fluviodeltaic channel deposits (Holdgate et al., 2001)or saceae source, which, according to the ATR-FTIR spectra of modern hydromorphic soils; (3) the palynological evidence shows abun- resins, is chemically indistinguishable from Araucariaceae (Tappert dant terrestrial spores and pollen, but a distinct lack of marine et al., 2011). Future studies utilising proton NMR would be microplankton (Christophel et al., 1987). Collectively, this evidence required to further differentiate between these possible sources rejects a coastal depositional environment for the Class II amber of (Lambert et al., 2010). Strahan and Anglesea Type A amber is the Eastern View Group. These probable autochthonous cadinene- believed to have originated from the same source, as evidenced based ambers raise the possibility that previous Class II amber by their highly similar ATR-FTIR spectra. The only significant devi- coastal occurrences at Cape York and South Australia were derived ation between the spectra remains the exomethylene peak at 887 from Australian fossil resin deposits rather than from sources in cmÀ1, which possess a significantly higher absorbance for Strahan Southeast Asia. Furthermore, these multiple findings of Class II Type A amber relative to Anglesea. Reduction in olefin concentra- ambers on the Australian mainland highlight palaeobiogeographic tion is a well-recognized indicator of amber maturity (Martínez- and/or chemotaxonomic inconsistencies regarding the accepted Richa et al., 2000; Moura and Zwick, 2001), suggesting that the producers of cadinene-based amber. Specifically, they suggest: amber from the Anglesea deposit possesses a higher maturity rel- (1) that Dipterocarpaceae (or mastixioid Cornaceae) were native ative to Strahan amber, despite being of a younger age. to Australia during the Eocene, despite their apparent absence from the fossil record; and/or (2) that a third, presently unidenti- 5.1.2. Type B fied botanical source was capable of exuding cadinene resins. The Amber from Anglesea described as Type B was found to corre- first option is unlikely, since the first occurrence of Diptero- spond closely to Group B (i.e. Class II) amber from Lambert and carpaceae is from lower Eocene deposits of India (Rust et al., Poinar (2002) and Lambert et al. (2015), as defined in solid state 2010; reviewed by Dutta et al., 2011), whilst the oldest fossils of 13C NMR with normal decoupling, by the absence of exomethylene probable affinity to the mastixioid Cornaceae are from the Late and carbonyl resonances and a highly similar aliphatic region shar- Cretaceous of Europe (Knobloch et al., 1993). By the Eocene, the ing five strong peaks at comparable resonances and intensities. mastixioid Cornaceae had gained a broad distribution, but only Specifically, Type B amber possessed a near identical 13C NMR across North America and Eurasia (see review by Manchester spectrum to Class II amber reported by Lambert et al. (2013) from et al., 2009)). Because of Australia’s geographic location at polar Kuala Tungkal, Jambi, Central Sumatra (Early Miocene). It also and subpolar southern latitudes from the Late Cretaceous to exhibited similar spectra to Class II amber reported by Lambert Eocene (Morley, 2003), the continent was effectively insulated and Poinar (2002) from the Hukawng Valley, Burma (Cenomanian; from terrestrial dispersal from India, Eurasia and North America age constraints by Cruickshank and Kob, 2003; Shi et al., 2012) and during this interval. the Claiborne Formation near Malvern, Arkansas (Eocene), differing only in the presence of a small peak at 15 ppm. Type B amber is 5.1.3. Type C also comparable to amber reported from Cape Paterson, Australia, Despite similarities with Type A amber in both NMR and ATR- which was originally reported as Early Cretaceous (Lambert et al., FTIR, Type C amber retained enough differences to warrant a sepa- 1993), but has since been identified as ex situ from a modern beach rate classification. Unlike types A and B, the ATR-FTIR and NMR spec- deposit in the area (Quinney et al., 2015). The ATR-FTIR spectra of tra of Type C amber was not immediately comparable to any Type B ambers possess many distinct features compared to Class Ib previously published spectrum (e.g., Lambert and Poinar, 2002; ambers of Type A, including a characteristically low carboxyl band Angelini and Bellintani, 2005; Tappert et al., 2011; Seyfullah et al., and a unique alkyl peak at 2952 cmÀ1. These same features are 2015). Type C amber does exhibit broad similarities to all Class I replicated in the ATR-FTIR spectra of other established Class II amber subclasses, particularly in the small peak at 30 ppm (charac- ambers, specifically amber from the Tadkeshwar lignite mine in teristic of classes Ia and Ib) and the spike near 35 ppm (characteristic western India (Dutta et al., 2009) and from Cape York, Australia of Class Ic; Lambert et al., 1993). However, these similarities are not (Colchester et al., 2006; Sonibare et al., 2014). This finding not only extensive enough to conclusively allow classification into any of the conclusively categorises Type B amber as Class II, but also suggests three subclasses. Further analysis using py-GC–MS would be that ATR-FTIR might serve as a relatively cheap, fast and accessible required to better classify Type C amber. diagnostic technique for distinguishing Class I (specifically Class Ib) It is possible that some of these spectral contrasts between and Class II amber. Class II, cadinene-based, resins are presently types C and A are a result of differences in taphonomy rather than only known to have been produced from two sources: Diptero- botanical source, as all Type C amber specimens exhibited exten- carpaceae (Anderson et al., 1992; Rust et al., 2010) and the ‘mastix- sive alteration textures. However, this possibility is countered by ioid’ Cornaceae (van Aarssen et al., 1994). The large majority of both the lack of chemical change between the altered extremities Class II amber, including all the deposits listed above, have been of Type C amber and unaltered core samples (see Section 5.2) attributed to Dipterocarpaceae, with cornaceous resins limited to and the lack of similar spectral features in altered amber at all the resin canals of fossil fruits in Germany and England (van other localities. Aarssen et al., 1994). However, Dipterocarpaceae is absent from both the macrofossil and pre-Quaternary pollen record 5.2. Taphonomy of amber at Anglesea and Strahan (Christophel, 1989), and modern biota (Ashton, 1982), of Australia. Previous studies on Australian Class II amber from the beaches of Four discrete alteration patterns were described in this study South Australia (Murray et al., 1994) and Cape York, northern Aus- (labelled as alteration patterns 1–4), each representing a different tralia (Sonibare et al., 2014), reconciled this by proposing that the pathway of degradation of amber classes I and II. amber was transported by ocean currents from a dipterocarpa- ceous source in Southeast Asia, although the source areas have 5.2.1. Alteration pattern 1 not yet been confirmed. This mechanism of deposition is consid- A major portion of altered Class Ib and all Type C amber anal- ered highly unlikely at Anglesea, the only locality of the present ysed in this study showed very minimal to no evidence of chemical study where Class II amber was found, for the following reasons: alteration from either NMR or ATR-FTIR. This was despite extensive (1) the thick A Group coal seams, from which all of the Anglesea physical alteration observed in each sample, including paling of Class II ambers were found, indicate prolonged intervals of stag- colour, widespread fragmentation and powdering and a lack of vit- nant water during deposition; (2) these deposits are interbedded reous surfaces. Physical degradation in the absence of chemical A.J. Coward et al. / Organic Geochemistry 118 (2018) 103–115 113 alteration has been reported previously by Waddington and Fenn consequence of the saponification of ester linkages formed (1988), who proposed that the crazing, crizzling and exfoliation between communol and communic acid, as mentioned above, of amber exposed to vapours of common air pollutants was due which would result in the restoration of primary alcohol functional to mechanical, rather than chemical, actions. This was thought to groups. be due to the rapid adsorption and desorption of the vapours caus- Pattern 2A amber is differentiated from pattern 2S by exhibiting a ing swelling and contraction in the amber structure. This process significant drop in exomethylene absorbance relative to unaltered could have been replicated by the exposure of amber to wildly fluc- amber from Anglesea, evident in both ATR-FTIR and NMR. The ther- tuating humidity levels, resulting in extensive crazing and crizzling mal oxidation of exomethylene bonds into carboxylic acids is a that may have advanced to the fine powdering that characterises recognised degradation pathway of Class I amber (Shashoua et al., the majority of Type A, pattern 1 amber. 2005; Pastorelli et al., 2012) and is a probable cause of the chemical differences between amber of patterns 2S and 2A. Note that a net loss 5.2.2. Alteration patterns 2S and 2A of carboxylic acid groups is still observed in pattern 2A amber, likely Occurring only in Class Ib samples, ambers with these patterns due to slower formation rates of carboxylic acids in comparison to were characterised by significant drops in carboxyl absorption rel- the formation and migration of metal carboxylates. ative to unaltered amber in both NMR and ATR-FTIR spectra. This observation stands contrary to many previous studies which have 5.2.3. Alteration pattern 3 found carboxyl concentration of amber during alteration to This pattern has only been identified in Class II amber from increase due to the oxidation of olefinic groups into carboxylic Anglesea, and remains unique as the only alteration pattern iden- acids (e.g., Williams et al., 1990; Shashoua et al., 2005; Pastorelli tified by this study to show a clear increase in carboxyl absorption. et al., 2012). However, a decrease of carboxylic acids in amber Deriving exact reaction pathways for Class II amber is difficult, has been reported at least twice in the literature, where it has been because the overall macromolecular structure of the amber is not attributed either to an unspecified maturation reaction (Lyons precisely known (Anderson and Muntean, 2000). Despite this, the et al., 2009), or the solubilisation and subsequent migration of increased concentration of carbonyl groups strongly suggests the monoterpenic and diterpenic salts, such as communate, that arise occurrence of oxidative processes. As observed in pattern 2S from the acid-base reactions between carboxylic acid groups and amber, oxidation commonly occurs in Class I amber by the reaction alkaline waters (Pastorelli et al., 2013a). With regards to matura- of exomethylene groups with oxygen to form carboxylic acids tion reactions, high temperature decarboxylation would be (Shashoua et al., 2005; Pastorelli et al., 2012). The slight reduction expected to apply equally to all amber in a given deposit, contra- in exomethylene groups of pattern 3, as interpreted from the ATR- dicting the localised variation in alteration observed at Anglesea. FTIR spectra, suggest a similar reaction may have occurred in some By contrast, the formation and mobilisation of metal carboxylates of the Class II amber present at Anglesea. Additionally, the decrease from the acid groups of communic acid and occluded, non- in alcohol concentration, evident in ATR-FTIR, suggests the occur- polymerised mono-and diterpenes is supported in the ATR-FTIR rence of a secondary reaction pathway involving the oxidation of spectra of patterns 2S and 2A amber by a broad increase of the alcohols into carboxylic acids. An analogous reaction has also been 1570 cmÀ1 absorbance band, previously assigned to the C–O bond speculated to occur in Class Ia amber through the thermal oxida- of metal carboxylates in amber (Khanijian et al., 2013). As it is the tion of communol into communic acid, although no direct evidence non-polymerised components of amber structure that are most of this has been obtained (Pastorelli, 2011a). likely to solubilise and be lost from the amber by this process, pat- The widespread oxidation of pattern 2A and 3 zones, all recov- tern 2S amber would be expected to retain the macromolecular ered from Anglesea, implies that many ambers of this deposit were polylabanoid framework and high cohesion of unaltered amber. exposed to the atmosphere at least once prior to collection. How- This is largely evident in the alteration textures of patterns 2S ever, Class II ambers lack any evidence of metal carboxylate forma- and 2A, with only a handful of zones exhibiting powdery and pow- tion and dissolution, a reaction pathway widespread at Anglesea in dery vitreous textures. This evidence suggests that some Class Ib the Class Ib ambers of pattern 2A. With two possible exceptions amber at Strahan and Anglesea may have been exposed to metal- (see pattern 4), Class II amber appears to remain resistant to the rich, alkaline waters either pre- or post-burial. A hypothetical source same degradation processes that define alteration pattern 2A, for this water is the partial dissolution of, and groundwater interac- which was widespread amongst Class Ib amber at the same tion with, the overlying carbonate units of the Torquay Group deposit. This is likely a consequence of the lower concentration (Holdgate et al., 2001). It is possible that exposure to an alkaline bur- of carboxylic acid and ester groups in Class II amber, as evidenced ial or pre-burial environment may have also facilitated the saponifi- in both the ATR-FTIR and NMR spectra, which would reduce the cation of esters within pattern 2 amber, specifically the ester bonds impact of metal-carboxylate formation and dissolution prevalent formed from the self-crosslinking of communol and communic acid in patterns 2S and 2A. This explains why, excepting minor colour in Class Ib amber (Poulin and Helwig, 2015). The saponification of changes from green to dark brown, almost all Class II specimens esters would again result in the formation and solubilisation of metal lack the extensive physical degradation, powdering and other carboxylates and the subsequent decrease in measured carboxyl non-vitreous alteration textures of altered Class Ib ambers. This concentration, as observed in pattern 2 amber. However, whilst observation highlights the importance of chemical structure and saponification has been reported to occur in Class Ia amber botanical source in dictating the specific reaction pathways of (Khanijian et al., 2013; Pastorelli et al., 2013a), Class Ib amber from amber alteration, as previously noted by Bisulca et al. (2012). Fur- Anglesea show no indication of ester loss in ATR-FTIR, expected to thermore, it suggests that certain amber types, especially those occur at 1720 cmÀ1 (Poulin and Helwig, 2015). As such, saponifica- with a high concentration of organic acids and similar functional tion is likely to only have played a small part in defining the overall groups, possess an inherently higher susceptibility to chemical chemistry of patterns 2S and 2A amber, if at all. degradation and would therefore remain proportionally underrep- Both patterns 2S and 2A amber also exhibited a substantial resented within the fossil record. increase in the absorbance of hydroxyl groups in FTIR. Increasing hydroxyl concentration can represent a decrease in the degree of 5.2.4. Alteration pattern 4 polymerisation in amber and resins (Tappert et al., 2011), suggest- The two zones categorised as alteration pattern 4 exhibited a ing that the polylabdanoid macromolecular structure of patterns broad similarity to the ATR-FTIR spectrum of unaltered Class II 2S and 2A amber may have begun to breakdown. This could be a amber from which they are both derived. Both spectra share a weak 114 A.J. Coward et al. / Organic Geochemistry 118 (2018) 103–115 carboxyl band and a strong alcohol absorbance, as well as a similar in either the accepted geographic distribution of Dipterocarpaceae exomethylene peak. However, pattern 4 amber exhibits substantial during the Eocene or in the established producers of Class II amber. differences in the primary methylene and methyl absorption bands, The complexity in structural composition of Anglesea and Stra- which have remained chemically stable across all other alteration han ambers has facilitated an extensive variety of physical and patterns. This suggests that the reactions influencing pattern 4 alter- chemical alteration that offers unique insights into the mecha- ation have impacted not only the functional groups, but the ter- nisms of amber degradation under geological and atmospheric penoid carbon chains as well. Introducing further complications, conditions. Zones of altered amber from Strahan and Anglesea the NMR spectrum of this alteration pattern displayed much sharper were shown to possess one of four discrete modes of alteration, peaks relative to any other spectrum, indicative of a well-defined, with the type of alteration predominately dependent on the speci- homogenous structure, a characteristic not consistent with the fic structural class of the amber specimen. Altered Class II ambers highly complex, molecular structure of amber. showed evidence of oxidation of exomethylene and alcohol func- No definitive conclusion can be made regarding the reaction tional groups into carboxylic acids and exhibited a close associa- pathways of pattern 4 amber. However, the strong absorbance of tion with a possible metal-carboxylate by-product. By contrast, the band at 1590 cmÀ1 indicates the likely presence of metal car- some altered, Class Ib ambers displayed chemical changes indica- boxylates within the material (Khanijian et al., 2013), suggesting tive of saponification and metal carboxylate formation, indicating a reaction pathway similar to that proposed for alteration patterns a prior exposure to alkaline waters. These findings show that expo- 2S and 2A. In this case, the removal of occluded, non-polymerised sure to oxygen and water can have detrimental impacts on the terpenoids by the formation of soluble metal carboxylates would physical and chemical integrity of amber deposits. However, have substantially altered the bulk carbon chemistry, potentially despite these processes, some palaeobiological information, such resulting in the wavenumber and intensity changes apparent in as amber class, was preserved. Additionally, a large population of the alkyl bands in ATR-FTIR. Furthermore, the solubilisation of altered Class Ib ambers did not show any significant chemical non-polymerised terpenoids would increase the homogeneity of alteration despite extensive physical degradation (e.g., concentric the remaining terpenoid polymers, potentially contributing in the discolouration, crazing, powdering, etc.). These results suggest unusually sharp peaks apparent in the NMR spectrum. However, valid biochemical data can still be retrieved from most altered this hypothesis is countered by the lack of any further evidence ambers. The substantial differences of both the type and extent of metal carboxylate formation in other Class II ambers. Alterna- of alteration in Class Ib and II ambers highlight the importance of tively, it is hypothesised that this material may represent an alter- botanical source in dictating alteration pathways and suggest an ation end-product of a yet unidentified reaction in Class II amber. underrepresentation in the fossil record of amber types possessing Indeed, the close association of the powder to the surfaces of large, a high susceptibility to alteration processes. amber fragments of alteration pattern 3 could imply a relationship with the oxidation reactions of that pattern. Acknowledgements

5.2.5. Impact on stored palaeobiological data The authors are grateful to Adele Pentland and Will de Silva for The consistent chemical stability of alteration pattern 1 ambers collecting many of the amber samples used in this study. Thanks implies that stored palaeobiological information, including botan- are also given to Finlay Shanks for assistance with the chemical ical source and stable isotope ratios, may remain preserved in a analysis of amber by ATR-FTIR, to Andrew Langendam for help significant proportion of altered, Class I amber. Nevertheless, as with the imaging of samples and to the Monash University Under- exhibited by alteration patterns 2 and 3, chemical degradation graduate Volunteer Program for their assistance with sample col- can still cause intensity changes to key spectral features that might lection and preparation. The authors are also indebted to the otherwise be used to distinguish amber class and botanical source. editor Sylvie Derenne and one anonymous reviewer whose insights This includes large intensity changes to carboxyl and exomethy- and recommendations greatly improved the manuscript. The field- lene peaks, which are important features for differentiating Class work and research of this study was supported by an Australian I and II amber in both FTIR and NMR spectra. However, some FTIR Research Council (ARC) Discovery Project grant (DP140102515) spectral differences between Class I (Type A) and II (Type B) amber awarded to JDS. remained unchanged by chemical alteration. These peaks are considered especially diagnostic of their respective class and are high- Appendix A. Supplementary material lighted in Fig. 3. These findings suggest that some palaeobiological information, such as amber class and botanical source, can still be Supplementary data associated with this article can be found, in retrieved from altered amber samples. the online version, at https://doi.org/10.1016/j.orggeochem.2017. 12.004. 6. Conclusions Associate Editor—Sylvie Derenne

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