Discussion
The reflectance of European butterflies followed the ecogeographical
patterns predicted by Bogert’s rule: butterfly species in colder regions
showed lower reflectance than species in warmer regions in both VIS and
NIR wavebands. This pattern was consistent for dorsal and ventral
reflectance of all body regions. The consistent pattern for both VIS and
NIR wavebands is not surprising because reflectance in these two parts
of the spectrum was highly correlated. However, even after removing the
effect of this correlation, residual NIR reflectance of the ventral
thorax-abdomen and basal wings still showed thermally adaptive patterns.
Thus, our results clearly demonstrate that thermal benefits drive
ecogeographical patterns of reflectance in European butterflies.
We also found evidence for Gloger’s rule in European butterflies. Delhey
proposed two different definitions of Gloger’s rule: a simple version
states that animals are darker in a more humid environments while a more
complex version includes differential effects of humidity and
temperature on different types of melanin pigments (Delhey 2019). Our
results generally follow the patterns predicted by the simple version of
Gloger’s rule: after accounting for the effect of temperature-related
variables (PC1), reflectance of most body regions was lower in species
found in a more humid area (i.e. with higher mean precipitation). The
trends were consistent for all body regions (coefficients of PC2 in
Tables 1 and S5-6 are all negative; although this relationship was not
statistically significant for the ventral thorax). This suggests that
not only thermal environments but the degree of humidity also affects
the ecogeographical patterns of butterfly reflectance. Camouflage in
warm and humid environments could drive this relationship because such
environments are usually covered by darker vegetations and under low
light conditions, favouring the occurrence of darker species (Xinget al. 2016; Cheng et al. 2018).
In accordance with a previous study on Australian butterflies (Munroet al. 2019), we found similar high correlations between VIS and
NIR reflectance. This is not surprising because reflectance varies
continuously and often gradually across the spectrum and the degree of
VIS reflectance generated by pigments, such as melanins, often correlate
with their NIR reflectance (Alla et al. 2009). However,
structural colour, which is common in butterflies, can produce a wide
diversity of spectral shapes with multiple peaks in different parts of
the spectrum, potentially enabling VIS and NIR properties to respond
differently to selection. Our results suggest that selection for thermal
benefits has shaped both VIS and NIR reflectance in European butterflies
because both showed patterns consistent with Gloger’s rule. However, the
ventral basal wing and thorax regions also showed thermally-adaptive
variation independent of their VIS reflectance. This implies that
butterfly reflectance might be tuned to modulate signalling or
camouflage needs in VIS reflectance and thermoregulatory needs in NIR
reflectance despite the constraints imposed by the correlations between
them (Munro et al. 2019).
Though entire wing reflectance also showed thermally adaptive
ecogeographical patterns, the strength of this relationship was weaker
than for the thorax and basal wing regions. The evolution of butterfly
reflectance is likely to be affected by multiple competing functions,
including camouflage and signalling (Silberglied 1984; Kapan 2001; Chenget al. 2018; van der Bijl et al. 2020). The stronger
climate-reflectance relationships for the thorax and basal wing area
suggest that the relative importance of thermoregulation is greater for
these body regions, consistent with their more critical role in
thermoregulation (Wasserthal 1983). Basal wing and thorax regions
comprise a smaller area than the entire wing and are pivotal for
thermoregulation due to haemolymph circulation and proximity to flight
muscles (Arnold 1964), thus they may be less affected by competing
selective pressures other than selection for thermal benefits.
Our results show that larger species have lower entire wing reflectance
than smaller species in the NIR but not VIS wavebands. In other words,
size correlates with NIR reflectance, but not colour. Why have larger
butterflies evolved lower NIR reflectance of the wings independent of
both climate and colour? Cryptic NIR adaptations of wings could
contribute to thermoregulation. Although heat transfer from the wings to
the thorax through conduction may be limited, heat transfer may be
greater for larger than smaller wings. Larger butterflies have been
shown to perform better at controlling temperature and affording
elevated body temperature (Gilchrist 1990; Bladon et al. 2020).
Alternatively, the wings may function as solar concentrators and reflect
the solar energy radiated across the entire wings towards the thorax
(Shanks et al. 2015). This effect should be greater for larger
species because larger wings can reflect more solar radiation towards
the thorax than smaller ones. Thus, larger species may have evolved
lower NIR reflectance than smaller species to modulate the amount of
reflective energy from the wings to the thorax. A third possibility is
that larger butterflies may prefer to be active in the shade and
crepuscular hours which could also drive the evolution of lower NIR
reflectance (Xing et al. 2016). The underlying reason for the
observed size-reflectance relationship remains to be tested.
Butterflies use both dorsal and ventral basking, and both dorsal and
ventral reflectance can contribute to the process of heat transfer,
depending on basking behaviour (Clench 1966; Kingsolver 1985). However,
ventral regions are additionally exposed during cooling down when
butterflies close their wings tightly to minimise the absorption of
solar radiation (Clench 1966). To avoid the absorption of unnecessary
heat during cooling down, it may be equally important to have high
ventral reflectance, especially for species in warmer climates. Thus,
the reflectance of ventral regions in butterflies may be a result of
evolutionary modulation between two conflicting selective pressures:
absorbing light energy when heating up and reflecting it when cooling
down. In cold climates, selection for low reflectance to enable rapid
warming may prevail, while in hot climates, there may be stronger
selection for high ventral reflectance to facilitate cooling. Indeed,
our results demonstrate that ventral surfaces had higher reflectance
than dorsal surfaces in most species and the difference was larger in
warmer climates. Notably this relationship was only present for the
thorax and basal wing regions that are crucial for thermoregulation.
This suggests that the evolution of the ventral surfaces of butterflies
is affected by thermoregulatory pressures related to both heating and
cooling.
Thermal benefits have been considered as one of the major selective
agents that operate on butterfly reflectance (Kingsolver 1988; Hegnaet al. 2013). Our findings provide the most comprehensive
evidence to date that climatic gradients have shaped both visible and
near-infrared reflectance of butterflies consistent with both Gloger’s
and Bogert’s rules. We also show that not all body regions were equally
affected, but the observed climate-reflectance relationship was stronger
for body regions that play a greater role in thermoregulation. This
highlights that the relative strength of competing selective pressures
(e.g. signalling, camouflage, heating up, or cooling down) may vary
between different body parts and these collectively have affected the
evolution of the reflectance properties of butterflies.