Discussion
In this study we tested for signatures of post-mating selection on
gamete traits in relation to population density, and possible
interactive effects of population density and sperm concentration on
sperm motility and fertilisation rates. Our study yielded four key
results: 1) males from high density populations produce smaller sperm
compared with males from low density populations, although we found no
difference in egg size between females from different population
densities; 2) females from low density populations have lower
fertilisation success, although this becomes less important as sperm
concentration increases; 3) variances in fertilisation success were
higher for females than males; and 4) gamete compatibility between males
and females increases as sperm concentrations increase.
We found that males from high density populations, where sperm
competition would be expected to be more prevalent (Evans and Lymbery
2020), produced smaller sperm compared to males from low density
populations. To the extent that sperm production trades-off against
sperm size (Parker 1982; Gomendio et al. 1991; Tourmente et al. 2011),
this result supports the prediction that an increase in the overall
magnitude of sperm competition should select for a greater number of
smaller sperm, while lower sperm competition risk should result in the
production of fewer longer sperm (Parker 1982; Stockley et al. 1997;
Gage and Morrow 2003; García-González and Simmons 2007; Lüpold et al.
2020). These results support comparative studies of fish that show sperm
length decreased with sperm competition risk (Stockley et al. 1997),
although some studies have found the opposite, with among species
comparisons showing a positive relationship between sperm competition
risk and sperm size (Gage 1994; Byrne et al. 2003; Lifjeld et al. 2010;
Johnson et al. 2013). The reason for such varying and contrasting
results likely arises from the complex relationship between sperm
morphology and sperm performance (e.g. swimming speed and/or
fertilisation potential). In some species (especially internally
fertilisation taxa) longer sperm may be associated with improved
swimming performance and provide a competitive advantage under sperm
competition (Gomendio et al. 1991; Parker et al. 2010; Johnson et al.
2013). However, sperm length may also be associated with sperm
longevity, with longer, larger sperm living longer than smaller sperm.
If so, producing longer-lived sperm may confer an important advantage in
broadcast spawners in low density populations, allowing more time for
sperm-egg encounters and increased fertilisation success (however see,
Levitan 2000). A study of the broadcast spawning tubewormGaleolaria gemineoa showed that sperm with small heads but long
tails were favoured in high-concentration environments, whereas sperm
with long heads were favoured at low concentrations and old ages
(Johnson et al. 2013). The longer sperm produced by males from low
density populations detected in this study may provide an advantage if
they have greater longevity, although longevity was not directly
assessed in this study. Our analysis also showed that longer sperm from
low density populations did not show greater motility or swimming
speeds, suggesting that the larger size did not confer a swimming
advantage. The relation between sperm size and fertilisation success
under sperm competition is complex and appears to be highly context
dependent and likely to covary with sperm swimming traits and longevity.
Theoretical and empirical studies predict that egg size should reflect
adult density, with females from low-density populations producing
larger eggs than their high-density counterparts, because larger eggs
present larger targets for searching sperm (Levitan 2006; Crean and
Marshall 2008; Evans and Lymbery 2020). In the present study we found no
difference in mean egg size between females from high- and low-density
populations. However, egg size is not the only mechanisms available to
females for increasing the effective target size of their eggs. For
example, it is well established that the eggs of many broadcast spawners
release sperm chemoattractants, which are thought to increase the
effective target size of eggs, thus making them more ‘visible’ to
searching sperm (e.g. for theory see Jantzen 2001). While we currently
lack explicit evidence that females can facultatively adjust the
production of sperm chemoattractants to match the fertilisation
environment, or that selection may favour increased production of such
attractants in populations with persistently low adult densities (and
therefore sperm limited), the idea of testing for differences either in
the composition or volume of sperm chemoattractants across populations
has merit. Such an adaptation may represent a more cost-effective
mechanism for increasing the target size of eggs compared to increasing
the structural size of eggs in sperm-limited environments and offers an
exciting area for future research.
We assessed fertilisation rates of females from low- and high-density
populations across a range of sperm concentrations. As population
densities decrease and the risk of sperm limitation increases, we
expected that females would produce eggs that are more readily
fertilised (i.e. greater fertilisation success at lower sperm
concentrations), while in high sperm density environments females should
increase ovum defences to reduce the risk of polyspermy (Frank 2000;
Firman and Simmons 2013; Kosman and Levitan 2014). Surprisingly, we
found no evidence to support these ideas. Indeed, we found that females
from high density sites produced eggs that were more readily fertilised
at lower sperm concentrations than eggs from females originating from
low density populations, although this difference became progressively
less apparent as sperm concentrations increased. Furthermore, our
finding that at the highest sperm concentration (2 ×
108 sperm ml-1), females from both
high- and low-density populations experienced similar magnitudes of
abnormally fertilised eggs fails to support the idea that females from
high density populations are better at blocking polyspermy compared to
females form low density populations (Kosman and Levitan 2014).
Interestingly, we found that the variance components for (normal)
fertilization rates across the range of sperm concentrations were
generally higher in females than in males, but also that variances for
females were initially high at lower sperm concentrations (2 ×
102 & 2 × 103ml-1), decreased when sperm concentrations were at an
intermediate level (2 × 104 & 2 ×
105 ml-1), before rising sharply
when sperm concentrations exceeded 2 × 106ml-1. Qualitatively similar patterns have been
reported in other broadcast spawning invertebrates (Levitan 2004),
suggesting that under sperm limitation, where average fertilisation
rates are low, there will be heightened opportunity for selection on
female traits that improve fertilisation rates. Under such conditions,
for example, we might expect stronger selection for increased egg size
and/or the heightened production of chemoattractants that increase
sperm-egg encounter rates (see Evans and Lymbery 2020). Under
intermediate sperm concentrations, fertilisation rates were generally
high (~80%) and the variance in normal fertilisation
rates was consequently very low. By contrast, despite high overall
fertilisation rates in the highest sperm concentration groups
(>2 x 106 ml-1), the
proportion of normally fertilised eggs decreased dramatically at high
sperm concentrations with a concomitant increase in the variance in
normally fertilised eggs (see Fig. 1a). Together these findings suggest
that the opportunity for selection on egg traits will depend critically
on local spawning conditions, and that such patterns may be reflected on
a broader spatial scale in divergent populations with varying adult
densities.
Our fertilisation assays indicated that as sperm concentration
increased, gametic compatibility effects became increasingly important.
This suggests that under sperm limited conditions, where the risk of
fertilisation failure is higher, any benefits associated with selecting
genetically compatible sperm (Kosman and Levitan 2014; Oliver and Evans
2014) are offset by the direct costs of leaving many eggs unfertilised.
However, as sperm concentration increases, eggs can afford to become
‘choosier’ as this will ensure that fertilisations are biased toward
genetically compatible sperm (Sherman et al. 2015; Lymbery et al. 2017)
whilst avoiding the direct costs associated with polyspermy. Although we
have yet to understand the mechanistic basis that underlie these
dynamics patterns of sperm-egg interaction, we suspect that sperm
chemoattraction and gamete surface proteins play important roles in
differentially regulating sperm-egg encounter rates across the sperm
concentration continuum (Evans and Sherman 2013). Irrespective of the
underlying mechanisms, our results provide further support to an
increasing number of studies that have shown that local environmental
conditions can influence the magnitude of compatibility effects between
male-female combinations (Levitan and Ferrell 2006; Levitan et al. 2007;
Nystrand et al. 2011; Lymbery and Evans 2013; Sherman et al. 2015;
Rudin-Bitterli et al. 2018). Collectively, these studies highlight the
importance of testing for compatibility effects across a range of
ecologically relevant environmental conditions.
In conclusion, our study revealed significant effects of population
density and sperm concentration on gamete morphology and fertilisation
dynamics, respectively. While the extent of variation at the population
level may be determined by both environmental and/or genetic factors,
the critical importance of gamete-level natural and sexual selection in
broadcast spawners (Evans and Sherman 2013; Evans and Lymbery 2020)
leads us to predict that much of the variation we observe in these
populations is adaptive. However, we also report highly dynamic patterns
of fertilisation across experimentally altered sperm environments,
highlighting the importance of phenotypic plasticity in governing
sperm-egg interactions and the likely dynamic selective environment in
which fertilisation plays out. We eagerly anticipate future work that
seeks to understand the extent to which the mechanisms underlying these
dynamic patterns of sperm-egg interaction are themselves plastic in
their expression.