Discussion
For understorey plants in temperate regions, leafing out and flowering
before canopy closure should be important for fitness (Ida and Kudo
2008, Augspurger 2008). Because the timing of these two events are often
developmentally and temporally correlated, selection on either event
cannot be accurately estimated independently (Lande and Arnold 1983,
Kelly 1992, Diggle 1999). We showed that early leaf-out was consistently
associated with early flowering in a population of L. vernus , and
that phenotypic selection was acting on both traits. The direction of
direct selection on leaf-out day differed among years, whereas direct
selection consistently favoured earlier flowering. Selection on leaf-out
day acted indirectly, via flowering time, in one of the three years.
Selection favoured shorter development times in two of three years.
There was no evidence for correlational selection on leaf-out and first
flowering day. Taken together, our results show that flowering phenology
is correlated with vegetative phenology during spring, and that
selection on vegetative phenology can affect selection on flowering
time. Still, in our study selection favoured early onset of flowering in
all three years study, also when accounting for indirect selection via
leaf-out day.
Leaf-out and first flowering day were significantly positively
correlated in all three years, implying that flowering time is
constrained by the timing of leaf-out in L. vernus , i.e.
individuals must start their vegetative development early in order to
flower early (cf. Diggle 1999, Sola and Ehrlén 2007). At the same time,
there was also considerable independent variation in the two traits.
Some of this variation might be attributed to differences in shoot
architecture, in terms of the placement of the first inflorescence on
the shoot relative to the first leaf (Diggle 1999, Sola and Ehrlén
2007). The moderately strong association between timing leaf-out and
flowering initiation found in this study is in accordance with the
results for other herb species (e.g. Kelly 1992, Dahlgren et al. 2007),
and suggests that selection can act independently on each trait, as well
as on relative timing.
We found direct phenotypic selection on leaf-out day in two of three
years. Interestingly, selection was in opposite directions in these two
years. Such among-year differences in the direction of selection might
be related changes in trait means due to plastic responses to
inter-annual variation in spring temperature. In our study, selection
favoured earlier leaf-out in the year when development in spring was on
average latest, but favoured later leaf-out in the year when average
spring development was fastest. This pattern could be the result of that
early leaf-out, relative to the population mean, implies a larger risk
in years when temperatures during early spring are higher and
development on average starts
earlier, and that the benefits of an early development are larger in
years when development on average starts later. Little is known about
the agents of selection on leaf-out time in plants, but for plants where
shoot development starts in early spring it is likely that weather
conditions, e.g. in terms of the timing of snowmelt or late frosts
events, constitute important agents of selection (cf. Inouye 2008,
Augspurger 2013). In the alpine shrub Salix herbacea , selection
favoured intermediate leaf-out time in sites with late snowmelt, and
early leaf-out in sites with early snowmelt (Sedlacek et al. 2015),
suggesting that variation in the direction of selection was mediated by
the local climate. Late spring frosts likely often mediate selection for
later spring development, as suggested by a study reporting that late
frost events in spring primarily damaged plants in later developmental
stages (Augspurger 2013). Selection on leaf-out could also be mediated
by seasonal variation in light availability, and individuals that
leaf-out early in spring before canopy closure are likely to have a
fitness advantage due to a longer period of resource acquisition.
Leafing out before canopy closure has been shown to be important for the
growth and survival of understorey tree saplings (Augspurger 2008).
Early spring phenology might also infer costs in terms of increased
herbivory (Roy et al. 2004, Sedlacek et al. 2015). In L. vernus ,
grazing is likely to be the most intense early in the season when there
are fewer alternative food sources, and it is possible that also
variation in grazing intensity among years contributed to the observed
variation in selection on vegetative phenology in our study (7% of
individuals were grazed in 2013, 26% in 2014 and 38% in 2015).
The consistent selection for early flowering found in this study is in
accordance with the results of previous studies with L. vernus ,
as well as with several other species (Harder and Johnson 2009,
Munguía-Rosas et al. 2011, Ehrlén and Valdés 2020). In our study, this
pattern persisted also after taking variation in vegetative phenology
into account. In addition, the results were similar for analyses that
relativized fitness and standardized phenology traits within vs. across
years (Table 1, Appendix S5). Selection for early flowering in L.
vernus has been found to be mediated by warm April temperatures (Ehrlén
and Valdés 2020). In many understorey species, early flowering is likely
advantageous because light availability. and possibly pollinator
activity, decrease rapidly as the canopy develops (Bertin and Sholes
1993, Ida and Kudo 2008, McKinney and Goodell 2010). This advantage is
likely to be particularly large under warm spring conditions. Taken
together, selection on both leaf-out and first flowering day in L.
vernus is likely driven by multiple biotic and abiotic factors that
vary in intensity and relative importance among years.
Although it has been suggested that selection on flowering time can be
mediated by correlated life-history traits, for example via correlations
between flowering initiation and flowering duration or between flowering
time and emergence time (Rathcke and Lacey 1985, Austen et al. 2017), we
are not aware of any previous study simultaneously estimating indirect
selection on flowering time and leaf-out day. In our study, there was
indirect selection for early leaf-out via start of flowering in 2014,
suggesting that early flowering initiation was a main benefit of early
leaf-out in that year. We found no statistical support for indirect
selection on flowering time acting via leaf-out day, and overall our
results suggest that observed consistent selection for early flowering
in L. vernus is not driven by indirect selection via the timing
of leaf-out. Still, selection for later leaf-out might to some extent
have counteracted and weakened selection for early flowering in one of
our study years; selection for early flowering in 2015 was less than
half the size of selection in the two other years. In that year,
selection for early flowering was not detectable without accounting for
effects of leaf-out day. This suggests that accounting for differences
in vegetative phenology can affect the ability to correctly estimate
selection on flowering time. However, rather than overestimating the
frequency of selection for early flowering, which we might have expected
given the previous literature (e.g. Rathcke and Lacey 1985, Austen et
al. 2017), neglecting variation in leaf-out time in our analyses would
have led to underestimation of the strength of selection for early
flowering.
In our study, phenotypic selection favoured short development times
between leaf-out and first flowering day in two of three study years. We
are unaware of any previous studies estimating selection on the time
period between leaf-out and flowering. However, it has been hypothesised
that rapid development to reproduction should be favoured since it
decreases the likelihood of damage before reproduction (Williams 1966,
Post et al. 2008). Still, the fitness effects of a short development
time between leaf-out day and first flowering day are difficult to
separate from the effects of early flowering, and a short development
time might simply be favoured because it allows for early reproduction.
It is also possible that selection for short development time is the
result of that the optimal leaf-out time is close to the optimal timing
of flowering initiation, and that the fitness benefits of a short
development time reflect independent benefits of leafing out and
flowering during a particular period.
While we found selection on the relative timing of leaf-out and first
flowering day in terms of development time, we found no evidence of
correlational selection on the two traits. That is, the benefits of a
shorter development time were independent of the timing of leaf-out and
flowering initiation. Estimates of correlational selection are overall
rare (Kingsolver et al. 2001), and for plants, we are aware of only one
study reporting significant correlational selection on combinations of
phenological traits (flowering time and fruit maturation, Kelly 1992).
To better understand how correlations among seasonal events affect the
selection on a focal phenological trait, we therefore need studies
estimating selection on combinations of seasonal events, as well as
correlations and indirect selection for such events.
Our analyses of phenotypic selection provide important information about
how selection can act on correlated life history traits. However, it is
important to remember that our findings were based on female
reproductive success and not on lifetime fitness, and it is possible
that selection acting via male fitness differs from selection via female
fitness. However, we consider it unlikely that including also male
fitness would substantially alter our estimates of selection on
phenological traits. Pollen is transferred between male and female parts
of flowers that have a similar phenology in L. vernus , and it is
likely that donating and recipient plants on average have a similar
phenology. It is also true that trade-offs between current and future
reproduction implies that some of the advantages of early flowering
observed in this study might be offset by reduced fitness in subsequent
years. Still, such costs of reproduction appear to be relatively modest
in L. vernus , and the probability of flowering is higher in
individuals that flowered in the previous year than individuals that
were non-reproductive (Ehrlén and Van Groenendael 2001). Lastly, to
predict evolutionary responses to observed selection, it is essential to
know to what extent observed trait variation correspond to genetically
based variation vs. plasticity. A previous study with L. vernusshowed that much of the variation in timing of flowering was plastic
(Fogelström and Ehrlén 2019), suggesting that observed phenotypic
selection might not correspond to genotypic selection and lead to
evolutionary responses.