Response to short-term experimental ambient solar radiation exclusion
We found a substantial difference in p CA between shaded and sun exposed Pinus sylvestris pollen grains (Table 3 and Fig. 3a, b). Shaded pollen produced 21% less p CA than the sun-exposed pollen, and the 95% credible interval for the differences of the two treatment means within each MCMC run did not cross zero, giving us strong statistical confidence in the observed difference between the two treatments (Fig. 3b). The individual trees show differences in how strongly they respond to the shading treatment; two trees barely have a reduction of p CA production whilst seven trees show a strong response to shading (Fig. 3c).
Discussion
The key finding in this paper is that p-coumaric acid in pollen grains responds to short-term changes in ambient solar radiation occurring in the last few-weeks to one-month prior to dehiscence. In the study investigating responses to natural variation in radiation between years, an average of 31% more p CA was produced in 2014 compared to 2013, with an equivalent difference in intensity of 29% in the equivalent pollen-development periods. In the ambient solar-radiation exclusion experiment the shade cloth was installed one month prior to pollen dehiscence (i.e., covering the pollen development period) and this resulted in a clear reduction (21%) in the amount of p CA. As the shading cloth treatment was conducted for individual branches within trees, this experiment also builds on previous preliminary evidence that this plastic response to short-term reduction in solar radiation can occur locally within the tree, at the cone bud level (Rozema et al., 2009). The inter-annual study was conducted across several Pinus species, and also suggests consistent species-specific differences above and beyond those explained by pollen-size variability alone.
These results support and enhance current understanding of pollen formation in Pinus spp. Although the pollen cone buds begin growing in August the previous year, the pollen grains within the bud start developing two to three months before dehiscence. During the last stages in pollen-grain development, the peritapetal membrane that contains sporopollenin (e.g. p CA) is produced and covers the exine (Dickinson and Bell, 1972, Rowley et al., 2000). The peritapetal membrane most likely plays an important role in production ofp CA, and this final stage happens less than 11 days before flowering (Rowley et al., 2000). We find that there was no difference in incoming solar radiation in Geneva during the bud growing period of 2013 and 2014, but there was a large difference in solar radiation during the pollen development period, in both one month and two months prior to flowering (1187 and 2762 W/m2, respectively, see Fig. 1). A short-term response within the pollen development period is thus the most likely way to explain the considerable difference in p CA between the two years.
Results from the inter-annual and inter-specific variability study also point to potentially important variation at the species level. For example, Pinus pinaster contained up to twice the amount ofp CA compared with the other species. One obvious explanation for these differences is pollen size. In general, P. pinaster pollen grains are approximately two times larger in biovolume than the other species. Indeed, when we correct for this, using Beug (1961)’s reported average pollen size, the difference between species is reduced (Fig. 2 d). This effect is most obvious for P. pinaster and P. nigra. However, after size is corrected for some species-level variation still remains, hinting at other potential genetic differences resulting in differences in p CA production or in response to solar radiation. However, in this study we were limited by the number of individuals found within the Geneva botanic garden. More individuals of each of the different species tested here would be required for more robust inferences around species effects.
These findings have implications for the usage of p CA as a chemical proxy in palaeoecological reconstructions. First, our findings indicate that the production of p CA is a plastic, short-term response to the environment, which implies that pollen grains from a given tree sampled across multiple years should accurately reflect changes in the local solar radiation signal during the dehiscence season. Second, pollen-chemistry measurements taken from sediment cores will represent an integrated early-seasonal radiation flux across the range of years represented in a given sediment sample. Depending on sedimentation rate and temporal resolution, a typical sample from the pollen record represents anywhere between 5 and 20 years, and any between-year environmental variation is therefore averaged out, giving us confidence that we can observe long-term trends (e.g. Willis et al. (2011)). Finally, the potential for species-level variation observed in our study implies that in sediment cores sampled in an area with known occurrence of several species of pine may make chemical reconstructions more complex. This is especially the case if these species vary greatly in pollen size (for example, in Spain Pinus nigra, P. pinasterand P. sylvestris have overlapping range areas (Debreczy et al., 2011). Given these complexities, we expect that applying size corrections to p CA ratios could improve the accuracy of the reconstruction of past solar radiation, especially if specific size-correction factors for a given site can be established.
One important consideration is that, in this study, we were unable to disentangle the possible effects of different parts of the incoming solar spectrum (e.g. UV-B, UV-A or PAR) on p CA production. For example, we were unable to obtain measurements for ground-based UV-B, UV-A or PAR intensities at the Geneva botanic garden, whilst the shading cloths in our solar-radiation exclusion experiment did not only filter UV-B radiation. Furthermore, temperature and humidity may have differed in the shading treatments in our exclusion experiment but our experimental design did not allow us to account for these potentially confounding factors.
However, despite these limitations, several lines of evidence suggest the most likely driver of the variations in p CA is variation in UV-B radiation. Firstly, experimental studies reveal increased sporopollenin-based p CA in response to increased UV-B radiation, also in experiments where PAR was kept constant (Blokker et al., 2006, Blokker et al., 2005, Rozema et al., 2001b). Second, increases inp CA have been observed across both latitudinal (Willis et al., 2011, Jardine et al., 2016) and elevational gradient studies (Watson et al., 2007, Lomax et al., 2012), but the relationship between UV-B and PAR is decoupled between elevation and latitude gradients (although in these broader-scale studies potential species-level effects remain difficult to disentangle). We suggest that further experimental studies, which focus on determining the quantitative dose-response relationship in Pinus pollen of p CA to different solar radiation wavelengths, are required.
In summary, we investigated the effect of short-term changes in ambient solar radiation on the production in UV-B absorbing phenolic acids (p- Coumaric acid; p CA) in Pinus spp. pollen, and also species-level variation. Our results in both studies show a plastic response in p CA production in Pinus spp. pollen due to short-term changes in early-season solar radiation. p CA content show strong species-level variability, largely reflecting differences in pollen size between species. Based on these underlying and species-specific plastic responses, we expect that the integrated signal obtained from pollen grains in sediment cores (i.e. pollen grains representative of multiple years) will be able to detect longer term changes of past solar radiation. Our findings thus support the usage ofp CA as a solar radiation proxy in palaeoecological reconstructions as an indicator of seasonal changes in solar radiation, and suggests directions for how the method can be refined, e.g., by exploring in more detail the timing of the response, the exact spectra of solar irradiance that drive the response, and sources and consequences of species-level variation.
Acknowledgements
We thank Linn C. Krüger for assistance with pollen-picking, Professor Daniel Jeanmonod for permission to sample pollen from Conservatoire et Jardin botaniques, Ville de Genève and H. John B. Birks for comments. The daily-mean ambient solar radiation data and daily-mean pine pollen dehiscence data have been provided by MeteoSwiss, the Swiss Federal Office of Meteorology and Climatology. Funding for this project was provided by the Norwegian Research Council FRIMEDBIO programme to the PARASOL project (Project number 214359). AWRS was also supported by the Norwegian Research Council FRIPRO project PollChem (Project number 249844).
Conflict of interest
The authors have no competing interests to declare.
Authors contribution
KJW and VV developed the “PAlynological Reconstructions of pAst SOLar radiation” (PARASOL) project, AWRS, MJ, and VV conceived the research idea and experimental design for this study, AWRS and MJ conducted the experiment, AWRS collected pollen from Bergen, MJ collected pollen from Geneva, MJ carried out the lab work, AWRS and MJ analysed the data, JC contributed on the statistical analyses, MJ wrote the paper, AWRS, KJW, and VV commented critically on drafts of the paper. All authors approved the final version.
Data availability statement
Data supporting the manuscript results were archived in the Dryad Digital Repository (https://doi.org/10.5061/dryad.7sqv9s4nv).
References
BELL, B. A., FLETCHER, W. J., RYAN, P., SEDDON, A. W. R., WOGELIUS, R. A. & ILMEN, R. 2018. UV-B-absorbing compounds in modern Cedrus atlantica pollen: The potential for a summer UV-B proxy for Northwest Africa. Holocene, 28, 1382-1394.
BEUG, H.-J. 1961. Leitfaden der Pollenbestimmung für Mitteleuropa und angrenzende Gebiete, Stuttgart, Gustav Fischer.
BLOKKER, P., BOELEN, P., BROEKMAN, R. & ROZEMA, J. 2006. The occurrence of p-coumaric acid and ferulic acid in fossil plant materials and their use as UV-proxy. Plant Ecology, 182, 197-207.
BLOKKER, P., YELOFF, D., BOELEN, P., BROEKMAN, R. A. & ROZEMA, J. 2005. Development of a proxy for past surface UV-B irradiation: A thermally assisted hydrolysis and methylation py-GC/MS method for the analysis of pollen and spores. Analytical Chemistry, 77, 6026-6031.
DEBRECZY, Z., RÁCZ, I. & MUSIAL, K. 2011. Conifers around the world : conifers of the temperate zones and adjacent regions : Vol. 1,Budapest, Dendro Press.
DICKINSON, H. G. & BELL, P. R. 1972. The role of the tapetum in the formation of sporopollenin-containing structures during microsporogenesis in Pinus banksiana. Planta, 107,205-215.
ELLISON, A. M. 2004. Bayesian inference in ecology. Ecology Letters, 7, 509-520.
FRASER, W. T., SEPHTON, M. A., WATSON, J. S., SELF, S., LOMAX, B. H., JAMES, D. I., WELLMAN, C. H., CALLAGHAN, T. V. & BEERLING, D. J. 2011. UV-B absorbing pigments in spores: biochemical responses to shade in a high-latitude birch forest and implications for sporopollenin-based proxies of past environmental change. Polar Research,30, 6.
GARRETT, G. & HADLEY, W. 2011. Dates and Times Made Easy with lubridate. Journal of Statistical Software, 40, 1-25.
JARDINE, P. E., FRASER, W. T., LOMAX, B. H., SEPHTON, M. A., SHANAHAN, T. M., MILLER, C. S. & GOSLING, W. D. 2016. Pollen and spores as biological recorders of past ultraviolet irradiance. Scientific Reports, 6, 39269.
JUNGFERMANN, C., AHLERS, F., GROTE, M., GUBATZ, S., STEUERNAGEL, S., THOM, I., WETZELS, G. & WIERMANN, R. 1997. Solution of sporopollenin and reaggregation of a sporopollenin-like material: A new approach in the sporopollenin research. Journal of Plant Physiology,151, 513-519.
LOMAX, B. H., FRASER, W. T., HARRINGTON, G., BLACKMORE, S., SEPHTON, M. A. & HARRIS, N. B. W. 2012. A novel palaeoaltimetry proxy based on spore and pollen wall chemistry. Earth and Planetary Science Letters, 353, 22-28.
LOMAX, B. H., FRASER, W. T., SEPHTON, M. A., CALLAGHAN, T. V., SELF, S., HARFOOT, M., PYLE, J. A., WELLMAN, C. H. & BEERLING, D. J. 2008. Plant spore walls as a record of long-term changes in ultraviolet-B radiation.Nature Geoscience, 1, 592-596.
MAGRI, D. 2011. Past UV-B flux from fossil pollen: prospects for climate, environment and evolution. New Phytologist,192, 310-312.
MAZZA, C. A., BOCCALANDRO, H. E., GIORDANO, C. V., BATTISTA, D., SCOPEL, A. L. & BALLARÉ, C. L. 2000. Functional Significance and Induction by Solar Radiation of Ultraviolet-Absorbing Sunscreens in Field-Grown Soybean Crops. Plant Physiology, 122, 117.
R CORE TEAM 2017. R: A language and environment for statistical computing Vienna, Austria: R Foundation for Statistical Computing.
ROWLEY, J. R., SKVARLA, J. J. & WALLES, B. 2000. Microsporogenesis inPinus sylvestris L. VIII. Tapetal and late pollen grain development.Plant Systematics and Evolution, 225, 201-224.
ROZEMA, J., BLOKKER, P., FUERTES, M. A. M. & BROEKMAN, R. 2009. UV-B absorbing compounds in present-day and fossil pollen, spores, cuticles, seed coats and wood: evaluation of a proxy for solar UV radiation.Photochemical & Photobiological Sciences, 8, 1233-1243.
ROZEMA, J., BROEKMAN, R. A., BLOKKER, P., MEIJKAMP, B. B., DE BAKKER, N., VAN DE STAAIJ, J., VAN BEEM, A., ARIESE, F. & KARS, S. M. 2001a. UV-B absorbance and UV-B absorbing compounds (para-coumaric acid) in pollen and sporopollenin: the perspective to track historic UV-B levels.Journal of Photochemistry and Photobiology B-Biology,62, 108-117.
ROZEMA, J., NOORDIJK, A. J., BROEKMAN, R. A., VAN BEEM, A., MEIJKAMP, B. M., DE BAKKER, N. V. J., VAN DE STAAIJ, J. W. M., STROETENGA, M., BOHNCKE, S. J. P., KONERT, M., KARS, S., PEAT, H., SMITH, R. I. L. & CONVEY, P. 2001b. (Poly)phenolic compounds in pollen and spores of Antarctic plants as indicators of solar UV-B – A new proxy for the reconstruction of past solar UV-B? Plant Ecology, 154,9-26.
ROZEMA, J., VAN GEEL, B., BJORN, L. O., LEAN, J. & MADRONICH, S. 2002. Paleoclimate: Toward solving the UV puzzle. Science,296, 1621-1622.
RSTUDIO TEAM 2015. RStudio: Integrated Development for R. RStudio, Inc., Boston, MA URL http://www.rstudio.com/.
SEDDON, A. W. R., FESTI, D., ROBSON, T. M. & ZIMMERMANN, B. 2019. Fossil pollen and spores as a tool for reconstructing ancient solar-ultraviolet irradiance received by plants: an assessment of prospects and challenges using proxy-system modelling.Photochemical & Photobiological Sciences, 18, 275-294.
SEDDON, A. W. R., JOKERUD, M., BARTH, T., BIRKS, H. J. B., KRÜGER, L. C., VANDVIK, V. & WILLIS, K. J. 2017. Improved quantification of UV-B absorbing compounds in Pinus sylvestris L. pollen grains using an internal standard methodology. Review of Palaeobotany and Palynology, 247, 97-104.
WATSON, J. S., SEPHTON, M. A., SEPHTON, S. V., SELF, S., FRASER, W. T., LOMAX, B. H., GILMOUR, I., WELLMAN, C. H. & BEERLING, D. J. 2007. Rapid determination of spore chemistry using thermochemolysis gas chromatography-mass spectrometry and micro-Fourier transform infrared spectroscopy. Photochemical & Photobiological Sciences,6, 689-694.
WEHLING, K., NIESTER, C., BOON, J. J., WILLEMSE, M. T. M. & WIERMANN, R. 1989. p-Coumaric acid - a monomer in the sporopollenin skeleton.Planta, 179, 376-380.
WICKHAM, H. 2009. ggplot2 : elegant graphics for data analysis. New York: Springer.
WICKHAM, H. 2016. scales: Scale Functions for Visualization. R package version 0.4.0, https://CRAN.R-project.org/package=scales.
WICKHAM, H. 2017. tidyverse: Easily Install and Load the ’Tidyverse’. R package version 1.2.1. https://CRAN.R-project.org/package=tidyverse.
WILKE, C. O. 2016. cowplot: Streamlined Plot Theme and Plot Annotations for ’ggplot2’. R package version 0.6.2,https://CRAN.R-project.org/package=cowplot.
WILLIS, K. J., FEURDEAN, A., BIRKS, H. J. B., BJUNE, A. E., BREMAN, E., BROEKMAN, R., GRYTNES, J. A., NEW, M., SINGARAYER, J. S. & ROZEMA, J. 2011. Quantification of UV-B flux through time using UV-B-absorbing compounds contained in fossil Pinus sporopollenin. New Phytologist, 192, 553-560.
Table 1. Number of species and number of individuals sampled in 2013 and 2014.