Environmental and genetic variation in phenotype
In this study we set out to explain the nature, origin, and maintenance
of phenotypic variation in L. minor in the field. Phenotype
varied widely among sites, with mean frond area varying by a factor of
two (Fig. 2A), and mean root length by a factor of more than eight (Fig.
2B). This variation was overwhelmingly the result of phenotypic
plasticity. Although there were persistent differences in phenotype
among sites in the common garden assay, the reduction of variation in
frond area by 93% and in root length by 96% (Fig. 3, Table 2) reveals
that among site phenotypic variation is almost exclusively
environmental. This is consistent with previous work that has shown a
large degree of plasticity in these traits, (Vasseur and Aarssen 1992,
Cedergreen and Madsen 2002) and the absence of local adaptation (Vámos
and van Moorsel 2022). Both among and within sites, the environmental
contribution to phenotypic variation was larger for root length than
frond area, which is also consistent with previous work reporting root
length as L. minor ’s most plastic trait (Vasseur and
Aarssen 1992). Phenotypic variation in L. minor in the field is
largely explained as a plastic response to the abiotic environment,
shifting its phenotype to levels of resource availability. 35% of among
site variation in frond area is explained by light availability, with
plants producing larger fronds in more heavily shaded environments. The
production of larger leaves in low light environments is a standard
ecophysiological response in plants (Meziane and Shipley 1999, 2001),
that influences fitness through photosynthesis, transpiration and
thermoregulation (Anten et al. 1995, Hirose et al. 1997). Similarly,
46% of among site variation in root length is explained by nutrient
availability with a dramatic increase for plants growing in sites with
low levels of dissolved N and P. This is consistent with previous
experimental work that has documented a plastic increase in root length
in L. minor in response to nutrient limitation (Cedergreen and
Madsen 2002). Although L. minor can uptake inorganic nutrients
through both the root and the frond (Landolt 1986, Cedergreen and Madsen
2002), this balance shifts depending on both nutrient availability
(Cedergreen and Madsen 2002), and irradiance (Cedergreen and Madsen
2004) with the production of longer roots resulting in an increase in
root N uptake and NO3 reduction. Variation in frond area
and root length in L. minor can be conceptualised as a simplified
root-shoot ratio (Cedergreen and Madsen 2002). A well-studied trait in
land plants (Brouwer 1962, Poorter and Nagel 2000), L. minorseems to respond to resource limitation by investing more biomass into
increasing the surface area of the tissue responsible for the uptake of
the limiting resource.
In addition to among-site phenotypic variation, we observed significant
phenotypic variation within sites. Whereas frond area varied
substantially both among sites and among microsites within sites, the
majority of variation in root length was at the among site level. Given
the largely environmental origin of this variation, it is perhaps
uprising that frond area would vary within sites due to the patch-like
variation in light availability caused by fine-scale shading from
macrophytes and riparian plants (Bell et al. 1991). In contrast, water
nutrient availability is likely much more homogenous within sites due to
mixing and diffusion resulting in most variation in root length
manifesting among and not within sites. For both frond area and root
length, the proportion of phenotypic variation with a genetic origin was
much higher within sites (26% and 21%) than among sites (7% and 4%).
The larger contribution of environmental variation to phenotype among
sites can be explained by the greater environmental variation at the
higher geographical resolution. However, we observed a surprisingly
large amount of within site genetic variation. Environmental variation
aside, the absolute amount of genetic variation in frond area was twice
as large within sites than among sites, and equal within and among sites
for root length. Whereas among site genetic variation is easily
explained by adaptation to local conditions or genetic drift given
limited gene flow, the large amount of within site genetic diversity is
surprising, especially in the absence of sexual reproduction.
In the common garden assay, the contribution of replicate flask to
overall phenotypic variation was significant and second only to residual
variation. This is perhaps surprising since replicate flasks consisted
of clones, descending from the same ancestor sampled from the field.
However, replicate flasks confounded several sources of variation
including flasks effect, chamber effect (from the blocked design), and
birth order effects from the original parental frond which have been
shown to persist over several generations (Barks and Laird 2015, 2016,
Mejbel and Simons 2018). Removing this variation from the residuals
enabled us to detect the higher-level effects of microsite and site.