Review of Palaeobotany and Palynology 221 (2015) 138–143

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Review of Palaeobotany and Palynology

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Nothofagus pollen grain size as a proxy for long-term climate change: An applied study on Eocene, Oligocene, and Miocene sediments from

Kathryn W. Griener ⁎,SophieWarny

Louisiana State University, Department of Geology and Geophysics, Baton Rouge, LA 70803, United States article info abstract

Article history: Understanding the nature of moisture availability in the Eocene, Oligocene, and Miocene of Antarctica is Received 17 February 2014 imperative for climate reconstructions and for understanding the potential influence of future climate change Received in revised form 6 June 2015 on modern landscapes. Previous studies have used traditional palynological techniques, fossil margins, Accepted 8 June 2015 hydrogen isotope leaf waxes, and palynomorph δ13C values to indicate changes in moisture on the southernmost Available online 20 June 2015 continent. Here, we explore the relationship between pollen width (equatorial diameter) and climate as a

Keywords: potential indicator of changes in moisture availability in Antarctica, a technique that had not been widely applied Nothofagus previously. To date, studies of pollen grain size response to environmental stress and the applicability of using Pollen fossil pollen grain size as a climate change proxy are limited. In this paper, we explore the relationship between Size pollen grain size and environmental stress (desiccation intensity) and whether such a relationship can be applied Climate to fossil pollen from Antarctica. We measured the widths of 157 modern specimens of Nothofagus spp. (the genus Antarctica of Southern ) pollen from throughout the Southern Hemisphere and compared them with mean annual Cenozoic precipitation records to assess the relationship between pollen grain size and moisture availability. The widths of 458 Antarctic Nothofagidites lachlaniae-complex pollen grains (from Eocene, Oligocene, and Miocene cores) were then measured and compared to other paleoenvironmental proxies to evaluate whether or not a significant trend in size variability can be associated with a change in moisture availability through geological time. Modern data show a significant relationship between decreased moisture availability and increased pollen grain size and are consistent with a previous study, providing confidence for using changes in pollen grain size as an indicator of moisture. Fossil data show a 23% increase in average pollen grain size from the late Eocene through the early to mid Miocene, indicating a decrease in moisture availability in Antarctica during this time. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Esjmond et al. (2011) hypothesized that pollen grain size and shape may affect pollen longevity through desiccation, and established a Pollen viability is the capacity of a pollen grain to “live, grow, germi- relationship between the pollen grain sizes of eight of the Rose nate or develop” (Lincoln et al., 1982), regardless of whether or not it family and desiccation stress. Principle component analysis of pollen undergoes germination. Pollen longevity is the period in which pollen grain size data empirically indicated that grain size increases under retains its viability, or germinability (Dafni and Firmage, 2000). Pollen desiccation stress for the three groups of pollen they analyzed (Rubus viability and longevity are dependent on several factors. Nepi et al. gracilis, Rosa,andCrategus; Esjmond et al., 2011). An increase in pollen (2001) regard the duration of pollen viability (i.e. pollen longevity) as grain size, corresponding with increased volume and a decrease in the being dependent on the potential for a grain to retain water while relative surface area to volume ratio, could increase the amount of maintaining its efficiency through intact plasma membranes, and time to pollen desiccation, thereby prolonging the viability of a grain, indicated that the viability of dehydrated pollen (as opposed to pollen i.e. increasing pollen longevity (Esjmond et al., 2011). with a naturally low water content) typically decreases with water It is therefore reasonable to consider that pollen grain size of at least loss (desiccation). some angiosperm species will change over time as a response to chang- es in humidity and/or moisture availability in an environment. Moisture availability in Antarctica is thought to have decreased since the Eocene, when temperatures were relatively high and the southernmost ⁎ Corresponding author at: E235 Howe-Russell Geoscience Complex, Louisiana State continent experienced mean annual precipitation levels greater than University, Baton Rouge, LA 70803, United States. Tel.: +1 985 246 9615; fax: +1 225 578 2302. 1000 mm/yr (e.g. Poole et al., 2005; Francis et al., 2008; Pross et al., E-mail addresses: [email protected], [email protected] (K.W. Griener). 2012; Griener et al., 2013), though a possible post-Eocene increase in

http://dx.doi.org/10.1016/j.revpalbo.2015.06.003 0034-6667/© 2015 Elsevier B.V. All rights reserved. K.W. Griener, S. Warny / Review of Palaeobotany and Palynology 221 (2015) 138–143 139 precipitation likely occurred around the Mid Miocene Climatic pers. comm.). Ten Oligocene samples were collected from the Optimum, as evidenced by hydrogen isotopes in leaf waxes (Feakins SHALDRILII 12A core. Diatom and calcareous nannofossil biostratigraph- et al., 2012). Palynological analyses conducted on cores from the south- ic age dating indicates an age between 28.4 and 23.3 Ma for this core ernmost continent indicate a deterioration of the climate through the (Anderson et al., 2011; Bohaty et al., 2011). Miocene sediments were late Eocene (e.g. Warny and Askin, 2011a) and into the Oligocene (e.g. collected from the ANDRILL 2A core from the Southern McMurdo Warny and Askin, 2011b) and Miocene, where several, short warming Sound in the Ross Sea. Ten samples from the lower section of the core intervals are apparent (e.g. Warny et al., 2009; Griener et al., 2015). (707.25–1139.5 mbsf) were used in this study. This section is dated at Many Antarctic palynological studies show a dominance of a single N20 Ma (1139.5 mbsf) to 17.235–17.533 Ma (707.25 mbsf) based on genus of terrestrial palynomorph, Nothofagidites spp., the genus of magnetostratigraphic data tied to bio- and isotope stratigraphy (Acton Southern beech (e.g. MacPhail and Truswell, 2004; Warny et al., 2006, et al., 2008). 2009; Anderson et al., 2011; Griener et al., 2015). This genus, which is extant throughout the Southern Hemisphere today in regions such as 2.3. Sample processing and data collection , , and , is thought to be sensitive to changes in water availability (e.g. Griener et al., 2013). We hypothesized Modern pollen grains were taken from herbaria flower specimens that Nothofagidites lachlaniae pollen grain size increased as Antarctica and did not require the use of hazardous chemical processing for extrac- became progressively drier from the late Eocene until the early to mid- tion from sediments; therefore, modern pollen grains were unpro- dle Miocene. (We note that, although many authors generally indicate cessed. These unprocessed modern specimens were mounted to pollen grain size to be fairly constant within a species and therefore a microscope slides using glycerine jelly. Fossil specimens were processed fi de ning characteristic, some variation does exist. For example, according to standard palynological techniques (e.g. Brown, 2008). Sed- Truswell and MacPhail (2009) saw a variation in Antarctic N. lachlaniae iment samples were treated with hydrochloric acid and hydrofluoric μ pollen that appears to range from ~25 to 32 m.) It is reasonable to acid to remove all calcareous and siliceous sediment material. Samples believe that N. lachlaniae pollen would experience an increase in grain were sieved at 6–100 μm fraction to remove additional inorganic size through time, increasing the volume to surface area ratio, in order material. The remaining palynomorph residue was mounted on to retain more water and prolong the time to desiccation. In this microscope slides using glycerine jelly. study, we measured the equatorial diameters (herein referred to as Modern and fossil Nothofagus pollen grain sizes were measured “ ” “ ” widths or grain size ) of pollen grains from nineteen modern using an Olympus BX40 microscope interfaced with QCapture Nothofagus spp. . We compared the relationship of grain microscope software. Modern specimens were identified to species size with known precipitation data from their respective growing level, and fossil specimens were identified to species or species- locations to assess the relationship between Nothofagus spp. grain size complex level during data collection. Each pollen grain specimen was and water availability. We then measured N. lachlaniae-complex photographed. At least one length measurement was taken for each of pollen grains from Eocene, Oligocene, and Miocene aged Antarctic the 157 modern and 458 fossil specimens. Although most species we Δ13 sediments and compared these data with palynological and C analyzed were fairly circular in polar view, multiple width measure- paleoenvironmental records. We hypothesize that increases in the size ments were taken on 22 modern specimens in order to assess any of pollen from the N. lachlaniae-complex will correlate with other potential intragrain width variations. Some modern pollen grains paleoenvironmental proxies that indicate a decrease in water availabil- showed obvious width variations. For consistency, we used only ity and that show an overall decrease in the abundance of N. lachlaniae measurements that were taken at the widest apparent transect for pollen through the Eocene, Oligocene, and Miocene, as the southern- comparison with precipitation data. most continent became drier.

2. Materials and methods 2.4. Data processing and analysis for modern samples

2.1. Modern specimens Multiple grains were measured from each herbarium Nothofagus to assess any intraplant variation in pollen size. The pollen grain Modern Nothofagus pollen grains were collected from 19 herbaria measurements were then averaged for each plant. To assess the rela- specimens obtained from the Harvard University Herbarium and the tionship between pollen grain width and water availability, we com- Herbarium at the Museum of New Zealand. The herbaria specimens pared average grain width with precipitation records. The collection were collected between 1907 and 1982 from Argentina, Chile, New year and approximate location for each plant specimen were obtained Caledonia, , New Zealand, and Papua (see Table S1 for via herbaria sheets, and monthly precipitation data for the collection latitudes, longitudes, and other collection data). The specimens year and location were accessed using the BIOCLIM database (Hijmans represent thirteen species of Nothofagus, and include members et al., 2005) and processed using ArcGis (Environmental Systems from all of the four Nothofagus subgenera (Brassospora, Fuscospora, Research Institute, Redlands, California, USA). MAP was calculated for Lophozonia,andNothofagus). each specimen using the obtained data and ranged from 829 mm/yr to 3778 mm/yr. The relationship between MAP and pollen grain width 2.2. Fossil specimens was assessed using ordinary least squares regression (OLS; Smith, 2009). Regression analysis was performed using PAST software Fossil specimens were collected from three Antarctic cores. The (Hammer et al., 2001). SHALDRIL NBP06-02A 3C (SHALDRILII 3C) and SHALDRIL NBP06-02A 12A (SHALDRILII 12A) cores were collected in 2006 from the James 2.5. Data processing and analysis for fossil samples Ross Basin of the Weddell Sea, off the coast of the . The SHALDRILII 3C core provided thirteen sediment samples from the For this study, we considered only fossil pollen grains assigned to the latest Eocene. A single strontium age date obtained for this core N. lachlaniae-complex in order to minimize any variation between indicates an age estimate of 35.9 ± 1.1 Ma (Bohaty et al., 2011), and is species and because the majority of fossil specimens were identified confirmed by diatom and dinoflagellate biostratigraphy, which indi- as belonging to this species-complex, thereby providing the largest cates a range in age between 37 and 33.7 Ma (Bohaty et al., 2011). possible fossil data set. Average pollen grain widths were calculated This core likely spans a short period (≤200 kyr), as evidenced by seismic for all N. lachlaniae specimens for each core depth. We compared fossil stratigraphy of the site and typical sedimentation rates (J.B. Anderson, pollen grain widths with palynological records and plant Δ13C values 140 K.W. Griener, S. Warny / Review of Palaeobotany and Palynology 221 (2015) 138–143 from the same cores in order to assess the validity of using fossil pollen grain size from reflecting changes in moisture availability, as long as as an indicator of paleoenvironmental climate change. the longest transect is consistently measured. Moreover, Antarctic fossil N. lachlaniae-complex specimens do not exhibit the variation in width 3. Results & discussion associated with the pollen grains from many of the species of the Brassospora group (see Fig. 2d–f). Pollen grains from the N. lachlaniae- 3.1. Modern specimens: evidence for pollen grain size as an indicator of complex exhibit minimal intragrain width variation (see Fig. 2g–i). moisture availability Interestingly, many modern Nothofagus species are anemophilous and pollen may therefore be exposed to harsh environmental conditions A total of 157 measurements of modern Nothofagus spp. pollen (i.e. aridity) after shedding or pollen dispersal (e.g. Báez et al., 2002). As widths were taken from the nineteen herbaria specimens. These pollen some Nothofagus species have been shown to be reproductively self- grains exhibited a wide range of widths: 16.52 to 34.88 μm for subgenus incompatible, cross-pollination is imperative for much Nothofagus Brassospora species; 23.36 to 34.89 μmforsubgenusFuscospora species; germination (e.g. Riveros et al., 1995, 1998; Palma et al., 1996). Palma and 23.95 to 36.50 μm for subgenus Nothofagus species. Only one pollen et al. (1996) estimated that the percentage of viable pollen that reaches grain was available for a species of the Lophozonia subgenus, with a the stigmas is as low as 47% in some modern Nothofagus species, and width of 34.58 μm (see Table S2). Intraplant variation in pollen grain Nothofagus pollen viability drops as low as 21% after six months of size was very small (b2 μm) for sixteen of the nineteen herbaria speci- storage (Báez et al., 2002). The anemophilous nature of Nothofagus,com- mens and ranged from 2.5 to 6.8 μm for the remaining three. Nothofagus bined with the need for reproductive cross-pollination, and apparent spp. pollen grain size exhibited a significant positive correlation with sensitivity to desiccation stress after shedding may explain the observed MAP (r2 = 0.66, n = 19, p = 0.0001; Fig. 1). When subgenera were response of Nothofagus pollen grain size to changes in moisture assessed separately, members of the Brassospora and Nothofagus availability presented here. subgenera, respectively, both showed significant positive correlations Sarkissian and Harder (2001) observed large and small Brassica rapa with MAP (r2 = 0.73, n = 9, p b 0.02 for Brassospora;r2 = 0.89, n = pollen under artificial selection over three generations. Third generation 4, p b 0.17 for Nothofagus), and members of the Fuscospora subgenus pollen from small-pollen lines of B. rapa decreased in size from their showed a positive correlation with MAP (r2 = 0.37, n = 5, p b .03). initial generation, and pollen from large-pollen lines increased through See Fig. S1. Specimens from the Brassospora group exhibited differences the generations (Sarkissian and Harder, 2001). The authors suggest a in intragrain widths (multiple widths from a single pollen grain) rang- scenario for the cause behind a heritable change in pollen size: if larger ing from 0.1 μmto9.4μm (average = 4.4 μm, n = 17). Specimens pollen grains of a species better contend for fertilization, pollen grain from the Fuscospora group exhibited differences in intragrain widths size will increase over time, as pollen size is heritable (Sarkissian and ranging from 0.1 μmto0.6μm (average = 0.2 μm, n = 6). Harder, 2001). Similarly, if larger Nothofagus spp. pollen grains are Our results from modern Nothofagus pollen are consistent with the better adapted to resisting desiccation after shedding, pollen grain size empirical results of Esjmond et al. (2011), who found that grains were will increase as a result of heritable pollen traits. Notably, Sarkissian larger under desiccation stress. These results provide confidence for and Harder (2001) observed a tradeoff between pollen diameter and using changes in Nothofagus spp. pollen grain size as a proxy for changes number of grains produced; small-pollen lines produced more pollen in moisture availability. The wide variation in intragrain widths exhibit- grains through the generations, while large-pollen lines produced ed by Brassospora group specimens is due to the morphology of these fewer grains. This trend is not linear, however, as the tradeoff will create pollen grains (see Fig. 2a–c). For consistency, only measurements a pollen size that balances the advantages of a larger average grain size taken at the widest point of these grains were considered for OLS with the disadvantage of reduced pollen production (Sarkissian and regression analysis, as these represent the maximum potential width Harder, 2001). The trend observed between modern Nothofagus spp. attained by these grains. Intragrain size variations of specimens from and MAP in our study may, therefore, plateau or flatten out as pollen the Fuscospora group were negligible (b0.6 μm). Regardless, our results grain size increases (i.e. pollen grain size will not continue to increase suggest that the variations in intragrain width do not prevent pollen unchecked at the expense of pollen production). Regardless, our modern data give confidence for using Nothofagus spp. pollen grain size as a proxy for moisture availability. 4000 r2 = 0.66; n = 19; p = 0.0001 Subgenus Brassospora 3.2. Fossil specimens: shifts in moisture availability in Antarctica in the Subgenus Fu scospora Subgenus Lophozonia Subgenus Nothofagus Eocene, Oligocene, and Miocene

3000 We present measurements from N. lachlaniae-complex specimens from Antarctic Eocene (SHALDRILII 3C), Oligocene (SHALDRILII 12A), and Miocene (ANDRILL 2A) cores. Eocene specimens ranged in size from 22.3 to 25.8 μm(average24.5μm; n = 13). Oligocene specimens μ μ 2000 ranged in size from 23.7 to 30.9 m (average 26.1 m; n = 10). Miocene specimens ranged in size from 26.6 to 36.4 μm (average 30.2 μm; n = 10). There is an overall increasing trend in pollen grain size from the Eocene through the Miocene (see Fig. 3). This represents a significant average size increase of 23% from the Eocene to the Miocene. 1000 Esjmond et al. (2011) suggest several factors that could complicate the application of this method to fossil specimens (i.e. using pollen grain size as a paleoenvironmental indicator). Each is discussed in detail below. (1) Any observed changes in average pollen grain size may be 0 due to changes in species assemblages (e.g. species with typically larger 10 15 20 25 30 35 grains may replace species with typically smaller grains over time, as a Mean Annual Precipitation at Specimen Collection Site (mm/yr) Average Grain Size (µm) result of environmental changes or other factors). Here, we examine the average grain sizes only for specimens assigned to the N. Fig. 1. Ordinary least squares (OLS) regression relationships between modern Nothofagus pollen size (μm) and mean annual precipitation (mm/yr). The dashed line indicates the lachlaniae-complex, one of the most common morphospecies observed linear regression through the modern data. in Antarctic fossil records (e.g. Warny et al., 2006; Truswell and K.W. Griener, S. Warny / Review of Palaeobotany and Palynology 221 (2015) 138–143 141

abc

de f

g h i

Fig. 2. Examples of modern and fossil Nothofagus specimens. Fig. 2a–faremodernNothofagus. Fig. 2g–iarefossilNothofagus specimens. Brassospora subgenus: 2a) N. rubra, EFS S28/1; 2b) N. starkenborghii,EFSK5/3;2c)N. discoidea, EFS V17/2. Fuscospora subgenus: 2d) N. grandis, EFS R16/3; 2e) N. solandri, EFS R26/3; 2f) N. fusca, EFS R27/3. Antarctic N. lachlaniae-complex specimens: 2g) ANDRILL 2 Core, 745.95 mbsf, EFS U36/3; 2h) SHALDRILLII 12A Core, 262 cmbsf, EFS O34/3; 2i) SHALDRILII 3C Core, 29 cmbsf, EFS R22/3.

MacPhail, 2009). Although some variability is observed in members of point out that sedimentary processes may contribute to reworking this species-complex, we believe that limiting the fossil dataset to the grains and grain size distributions. Warny and Askin (2011a, 2011b) N. lachlaniae-complex is appropriate, as it excludes any morphologically observed primarily in situ pollen grains in the SHALDRILII 3C (Eocene) and physiologically dissimilar species. (2) Esjmond et al. (2011) also and SHALDRILII 12A (Oligocene) cores. Moreover, grains that exhibited

Miocene Samples Oligocene Samples Eocene Samples (ANDRILL 2) (SHALDRIL 12A) (SHALDRIL 3C)

35

30

25 Complex Grain Size (µm)

Average Nothofagidites lachlaniae 20 cab 700 800 9001000 1100 5004003002001000 300 13001100900700500 Core Depth (mbsf) Core Depth (cmbsf) Core Depth (cmbsf)

Fig. 3. Change in average Nothofagidites lachlaniae-complex grain size through time. a) Average sizes of N. lachlaniae-complex specimens in the SHALDRIL 3C Eocene Core. The dashed line indicates a strontium age date of 35.9 Ma (Bohaty et al., 2011). Measurements of fossil pollen are reflected in μm, and depth below sea floor is measured in cmbsf. b) Average sizes of N. lachlaniae-complex specimens in the SHALDRIL 12A Oligocene Core. Measurements of fossil pollen are reflected in μm, and depth below sea floor is measured in cmbsf. c.) Average sizes of N. lachlaniae-complex specimens in the ANDRILL 2 Miocene core. The dotted line represents an age date of 17.533 Ma, and the dashed line represents an age date of 18.524Ma (Acton et al., 2008). Measurements of fossil pollen are reflected in μm, and depth below sea floor is measured in mbsf. 142 K.W. Griener, S. Warny / Review of Palaeobotany and Palynology 221 (2015) 138–143 obvious signs of potential reworking (e.g. thermal alteration, tearing, Acknowledgments etc.) in the SHALDRILII 3C, 12A, and ANDRILL 2A cores were excluded from analysis. (3) Lastly, Esjmond et al. (2011) point out that sedimen- We thank the Herbarium at the Museum of New Zealand and the tary processes may reflect temporal climate variations. We accept this Harvard University Herbarium for providing modern Nothofagus premise, but argue that assessing broad temporal climatological shifts specimens and J. Ian Raine, Lowell Urbatsch, and Jennifer Kluse for provides valuable information about climate change over long periods assistance in obtaining herbaria material. Thanks to the Antarctic of time (i.e. millions of years). Research Facility at the University of Florida for core sampling and Our results indicate an overall decrease in moisture availability in SHALDRIL and ANDRILL research teams for core collection. Funding for Antarctica from the Eocene through the Miocene. We consider several this project was provided through the NSF CAREER program (Grant potential causes behind the observed change in moisture availability #1048343 to SW) and through the Louisiana State University Marathon experienced in Antarctica over the Eocene, Oligocene, and Miocene. GeoDE Fellowship (Grant #101072 to KWG). Additional thanks are Several studies give evidence for a decrease in precipitation amount extended to Daniel Buechmann, who helped access precipitation data from the Eocene to the Miocene (e.g. Warny et al., 2009; Pross et al., in the BIOCLIM package using GIS software. Thanks are also extended 2012; Passchier et al., 2013). Increasing glaciation on the southernmost to Rosie Askin for assistance with Nothofagidites classification. continent during this period (e.g. Kirshner and Anderson, 2011; Warny and Askin, 2011a,2011b) may have contributed to a decline in moisture References availability. Additionally, factors such as atmospheric circulation pattern shifts or orbital forcing could play a role (see Griener et al. (2013) and Acton, G., Crampton, J., Di Vincenzo, G., Fielding, C.R., Florindo, F., Hannah, M., Harwood, references therein for a discussion of the potential factors behind D.M., Ishman, S., Johnson, K., Jovane, L., Levy, R., Lum, B., Marcano, M.C., Mukasa, S., Ohneiser, C., Olney, M.P., Riesselman, C., Sagnotti, L., Stefano, C., Strada, E., Taviani, changes in moisture availability in the Antarctic). M., Tuzzi, E., Verosub, K.L., Wilson, G.S., Zattin, M., ANDRILL-SMS Science Team, 13 Griener et al. (2013) found that Δ C values of SHALDRILII 3C 2008. Preliminary integrated chronostratigraphy of the Site AND-2A, ANDRILL Nothofagidites fusca group specimens decreased throughout the late Southern McMurdo Sound Project, Antarctica. In: Harwood, D.M., Florindo, F., Talarico, F., Levy, R.H. (Eds.), Studies from the ANDRILL Southern McMurdo Sound Eocene, indicating a decrease in moisture availability. Pollen grain Project, Antarctica. Terra Antartica. 15, pp. 211–220. measurements from the same core (presented here) generally correlate Anderson, J.M., Warny, S., Askin, R.A., Wellner, J.S., Bohaty, S.M., Kirshner, A.E., Livsey, D.N., to the Δ13CvaluesfromGriener et al. (2013);aspollengrainsize Simms, A.R., Smith, T.R., Ehrmann, W., Lawver, L.A., Barbeau, D., Wise, S.W., Kulhenek, increases, pollen Δ13C decreases in the SHALDRILII 3C cores, indicating D.K., Weaver, F.M., Majewski, W., 2011. Progressive Cenozoic cooling and the demise of Antarctica's last refugium. Proc. Natl. Acad. Sci. 108, 11299–11726. that the two separate environmental proxies give similar results. For Báez, P., Riveros, M., Lehnebach, C., 2002. Viability and longevity of pollen of Nothofagus the most part, increases in pollen grain size coincided with decreases species in South Chile. N. Z. J. Bot. 40, 671–678. in palynological counts of N. lachlaniae from the same cores (Warny Bohaty, S.M., Kulhanek, D.K., Wise Jr., S.W., Jemison, K., Warny, S., Sjunneskog, C., 2011. Age assessment of Eocene– drill cores recovered during the SHALDRIL II and Askin, 2011a,2011b; Griener et al., 2015). This observation is consis- expedition, Antarctic Peninsula. In: Anderson, J.B., Wellner, J.S. (Eds.), Tectonic, tent with those of Sarkissian and Harder (2001) who noted the tradeoff Climatic, and Cryospheric Evolution of the Antarctic Peninsula. Am. Geophys. between pollen size and pollen production discussed above. Together, Union, Washington, D.C., pp. 63–114. Brown, C.A., 2008. Palynological techniques. In: Riding, J.B., Warny, S. (Eds.), Second these results provide further evidence that overall changes in pollen EditionAm. Assoc. Strat. Palynologists. grain size reflect changes in moisture availability in Antarctica and, Dafni, A., Firmage, D., 2000. Pollen viability and longevity: practical, ecological and perhaps more importantly, that observing changes in pollen grain size evolutionary implications. Plant Syst. Evol. 222, 113–132. Esjmond, M.J., Wrońka-Pilarek, D., Esjmond, A., Dragosz-Kluska, D., Karpińksa-Kołaczek, presents a viable tool for studying changes in moisture availability. M., Kołaczek, P., Kozłowski, J., 2011. Does climate affect pollen morphology? Optimal size and shape of pollen grains under various desiccation intensity. Ecosphere 2, 1–15. 4. Conclusions Feakins, S.J., Warny, S., Lee, J.-E., 2012. Hydrologic cycling over Antarctica during the middle Miocene warming. Nat. Geosci. 5, 557–560. Francis, J.E., Ashworth, A., Cantrill, D.J., Crame, J.A., Stephens, R., Tosolini, A.-M., Thorn, V., Antarctic paleoclimate research presents a unique set of challenges, 2008. 100 million years of Antarctic climate evolution: evidence from fossil plants. In: such as the difficulty and expense of traveling to the southernmost Cooper, A.K., Barrett, P.J., Stagg, H., Storey, B., Stumb, E., Wise, W., 10th ISAES editorial continent, obtaining cores below ice-sheets, and the scarcity of exposed team (Eds.), Antarctica: A Keystone in a Changing World. Proceedings of the 10th Intl. Symp. Antarctic Earth Sci., Washington, D.C.Antarctic Earth Sci., Washington, D.C., and accessible outcrops. In addition to standard palynological techniques pp. 19–27 (e.g. MacPhail and Truswell, 2004; Warny et al., 2009; Warny and Askin, Griener, K.W., Nelson, D.M., Warny, S., 2013. Declining moisture availability on the Antarctic Peninsula during the late Eocene. Palaeogeogr. Palaeoclimatol. Palaeoecol. 2011a,2011b), organic geochemical methods have been applied to – – fi 383 384, 72 78. Antarctic paleoclimate studies, speci cally to understanding changes Griener, K.W., Warny, S., Askin, R., Acton, G., 2015. Early to middle Miocene vegetation in moisture (e.g. Feakins et al., 2012; Griener et al., 2013). However, history of Antarctica supports eccentricity-paced warming intervals during the these techniques are often time consuming, require the use of special- Antarctic icehouse phase. Glob. Planet. Chang. 127, 67–78. Hammer, Ø., Harper, D.A.T., Ryan, P.D., 2001. PAST: paleontological statistics software ized laboratories, and may contain inherent restrictions (for example, package for education and data analysis. Palaeontol. Electron. 4, 9. Kohn (2010) showed that the relationship between water and plant Hijmans, R.J., Cameron, S.E., Parra, J.L., Jones, P.G., Jarvis, A., 2005. Very high resolution δ13C may only be linear when MAP b ~1500 mm/yr). Therefore, the de- interpolated climate surfaces for global land areas. Int. J. Climatol. 25, 1965–1978. Kirshner, A., Anderson, J.B., 2011. 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