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).
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Table 1. Number of species and number of individuals sampled in
2013 and 2014.