1 Supporting Information Appendix 2 Pliocene reversal of late Neogene aridification 3 4 J.M.K. Sniderman, J. Woodhead, J. Hellstrom, G.J. Jordan, R.N. Drysdale, J.J. Tyler, N. 5 Porch 6 7 8 SUPPLEMENTARY MATERIALS AND METHODS 9 10 Pollen analysis. We attempted to extract fossil pollen from 81 speleothems collected from 11 16 caves from the Western Australian portion of the Nullarbor Plain. Nullarbor speleothems 12 and caves are essentially “fossil” features that appear to have been preserved by very slow 13 rates of landscape change in a semi-arid landscape. Sample collection targeted fallen, well 14 preserved speleothems in multiple caves. U-Pb dates of these speleothems (Table S3) 15 ranged from late Miocene (8.19 Ma) to Middle Pleistocene (0.41 Ma), with an average age 16 of 4.11 Ma. 17 Fossil pollen typically is present in speleothems in very low concentrations, so pollen 18 processing techniques were developed to minimize contamination by modern pollen (1), but 19 also to maximize recovery, to accommodate the highly variable organic matter content of the 20 speleothems, and to remove a clay- to fine silt-sized mineral fraction present in many 21 samples, which was resistant to cold HF and which can become electrostatically attracted to 22 pollen grains, inhibiting their identification. Stalagmite and flowstone samples of 30-200 g 23 mass were first cut on a diamond rock saw in order to remove any obviously porous material. 24 All subsequent physical and chemical processes were carried out within a HEPA-filtered 25 exhausting clean air cabinet in an ISO Class 7 clean room. Samples were washed repeatedly 26 in distilled, deionized water, then etched in dilute (c. 0.5 M) HCl for 10 minutes to remove 27 the outer few 100 µm of calcite, before washing repeatedly again in distilled water. Exotic 28 Lycopodium spores (available in tablet form from Lund University, Sweden) were added to 29 each sample in order to allow pollen concentrations to be estimated and to provide bulk for 1 30 centrifugation to act upon, before samples were dissolved in concentrated (12 M) HCl. The 31 CaCl-rich solute was then diluted approximately four-fold with distilled water before 32 centrifugation (3500 ppm for 3 minutes) to discard the solute component. Organic fractions 33 were separated from residues using a Na-polytungstate heavy liquid (2) (empirical formula 34 Na6O39W12) made up to specific gravity 2.0. Those samples which yielded a substantial 35 organic-rich supernatant were then immersed in 10% KOH at 80°C for 2-10 minutes. 36 Following KOH, these samples, along with samples that yielded minimal organic 37 supernatants, were dehydrated in 100% ethanol, then suspended in glycerol. Acetolysis was 38 avoided in order to aid the identification of modern pollen contaminants (1). 39 The majority of speleothems contained ≤0.1 pollen grains g-1, and, for most samples, 40 no pollen was observed during complete scans of multiple glass slides representing ≥50% of 41 the acid-resistant residue. Statistically meaningful quantities of pollen were recovered from 42 13 speleothems, mostly flowstones, from five caves (Table 1). We initially incorporated 43 Carrera marble blanks into our processing protocol in order to monitor contamination rates, 44 but it quickly became clear that the majority of samples in any processing batch contained no 45 fossil pollen. The 68 (84% of the total) processed speleothems that contained no detectable 46 fossil pollen thus served as analytical blanks which provided an indication of the rate of 47 contamination by modern pollen. In total, six obviously modern (retaining cell contents and 48 in perfect condition) pollen grains (of four taxa, Ulmus, Alnus, Plantago lanceolata type, and 49 Poaceae) were observed in six of the 81 residues, implying an extremely low rate of 50 contamination, relative to the total number of fossil pollen grains, commensurate with our 51 use of HEPA-filtered workstations for this work. The first three modern pollen types 52 represent exotic taxa present in urban environments near our Melbourne laboratory, and 53 easily identified if encountered in a polleniferous sample. Poaceae includes many native 54 species, so modern contaminant grains conceivably could be mistaken for fossils. However, 55 the observed rate of pollen contamination implies that the proportions of fossil Poaceae, 0- 2 56 3.3% of the pollen sum, might in reality be as much as 0.2% lower, or 0-3.1% of the sum, a 57 small difference that does not affect our interpretation of late Neogene vegetation and 58 climate. In order to assess the possibility that speleothem pollen was derived from the host 59 limestone, we processed samples of the Early-Mid Miocene Nullarbor Limestone. No pollen 60 grains were observed in these residues. 61 Where possible, >100 pollen grains were counted from each sample. To achieve this in 62 some cases several aliquots of speleothem were dissolved, up to 760 g, and in most cases the 63 entire acid- and alkali-resistant residue was examined. Pollen was counted under compound 64 light microscopy at 640 and 1600 x magnification on a Zeiss Axiolab A1 with N-Achroplan 65 objectives, and photographed on a Zeiss Axioscope A1 compound microscope with EC Plan- 66 Neofluar objectives. All identified pollen types were included in pollen sums. The size of 67 pollen sums unavoidably varied by approximately one order of magnitude, which entailed 68 large variability in confidence estimates of pollen percentages (3). We therefore computed 69 95% confidence intervals for observed pollen percentages (3, 4) in order to explicitly 70 account for this variability. Deteriorated and unidentifiable grains were observed in low 71 quantities, but generally pollen was either in acceptable to excellent condition, or absent. 72 Pollen and spore identifications were made by comparison with pollen reference 73 collections and with published pollen atlases, including the Australasian Pollen and Spore 74 Atlas (apsa.anu.edu.au). For taxonomic groups for which existing knowledge of pollen 75 morphological diversity was poor, new modern pollen reference material was generated by 76 sampling voucher specimens at the National Herbarium of Victoria (MEL), supported by 77 new collections in the field. For Banksia (Proteaceae), which includes approximately 170 78 species, we took advantage of the availability of a nearly complete species level molecular 79 phylogeny based on five chloroplast DNA regions (5-7) to map pollen morphological 80 features observed under light microscopy onto a phylogenetic framework. Below, pollen 3 81 nomenclature follows Punt et al. (8) and updates at http://www.pollen.mtu.edu/glos-gtx/glos- 82 int.htm. 83 Speleothem pollen taphonomy. Below, we briefly summarize current understanding of 84 modes of pollen transport into caves, before considering the role of taphonomic processes in 85 the transport and preservation of pollen in Nullarbor speleothems. Although rates of pollen 86 deposition in caves are typically much less than in lake sediments, studies indicate that cave 87 sediment pollen spectra are representative of the surrounding vegetation (9), and similar to 88 the spectra found in nearby outdoor pollen traps and small lakes (9, 10). Pollen may be 89 brought into caves within drip water, through airborne transport, by animals, or by streams 90 (11). The status of drip waters as pollen sources is equivocal, with some studies finding 91 substantial quantities of pollen in drip water (12), others finding very little or none (10, 11). 92 Studies of airborne pollen transport and deposition within caves indicate that the quantities 93 of deposited pollen decrease with distance into the cave (9, 10). Where pollen is transported 94 into caves by animals, its impact on pollen spectra is expected to increase with cave depth, as 95 airborne pollen fluxes decline (13, 14). 96 The Nullarbor Plain is a broad surface of low relief, with limited deformation of its 97 horizontally oriented stratigraphy since its emergence from the sea during the mid-late 98 Miocene (15). There are no known lithological sources of reworked pollen on the Nullarbor 99 Plain other than the Nullarbor Limestone itself (which contained no pollen, see above). 100 Speleothems were collected from caves at depths below surface of 10-20 m. Drip water flow 101 paths are interpreted to be relatively simple through the overlying epikarst. Modern entrances 102 to late Neogene Nullarbor caves are unlikely to have persisted in the landscape for more than 103 a few 100 kyr, and are uninformative about the location of cave entrances at the time of 104 speleothem growth during the late Neogene. Thus, we do not know the position of any 105 speleothem with respect to cave openings at the time of its growth. 4 106 The acid-resistant residues of approximately 60% of the 81 processed speleothem 107 samples contained only small amounts of quartz and/or other siliciclasts. The remaining 108 ~40% included a silt- to clay-sized biogenic and mineral fraction comprising fungal spores, 109 charcoal and heavy minerals, which we interpret as airborne dust. Of these, 13 samples 110 included usable concentrations of pollen. The simplest interpretation of this pattern is that 111 the ‘clean’ Nullarbor speleothems grew at times when their caves had no connection to the 112 surface, while those containing biogenic/mineral dust fractions grew at times when their 113 caves had one or more entrances open to the atmosphere, allowing dust to be introduced onto 114 growing speleothem surfaces. Because the polleniferous samples represent a subset of 115 samples containing biogenic and mineral dusts, we infer that pollen was not generally 116 derived from drip waters, but from the atmosphere. This interpretation implies that, at least 117 for Nullarbor caves, the presence of fossil pollen within speleothems depends both on a cave 118 having had ≥1 entrances at the time of speleothem growth, and on other conditions at the 119 speleothem surface having being favourable for pollen preservation.