1 INTRODUCTION
Pollination mutualism is one of the major interaction systems between
plants and animals, and through this interaction, flower visitors
contribute to the reproduction of plants in different ways (Galen, 1996;
Scobell & Scott, 2002; Herrera et al., 2006; Inoue et al., 2007; Gómez
et al., 2009; Dohzono & Suzuki, 2010; Nattero et al., 2011). Adaptation
to locally different pollinator assemblages within the distribution
range of a plant species leads to local morphological specialization,
which may cause trait diversification and speciation in the plants
(Grant & Grant, 1965; Stebbins, 1970; Galen & Newport, 1987).
Geographic variation in floral traits such as flower size and shape
(Gómez et al., 2006; Nagano et al., 2014), corolla tube size (Hodges,
1997; Fenster et al., 2004), odor (Pellmyr, 1986; Majetic et al., 2009),
and color (Campbell et al., 1997; Newman et al., 2012) are considered to
have evolved as a local adaptation to regional pollinators. In
particular, morphological matching between floral spur length and
pollinator proboscis length is well known, with the textbook example
being Darwin’s hawkmoth and orchid (Darwin, 1877; Nilsson, 1988). In
fact, geographic correlations between floral size and pollinator size
have been reported in a variety of plant taxa (Alexandersson & Johnson,
2002; Herrera et al., 2006; Anderson & Johnson, 2008; Johnson &
Anderson, 2010; Boberg et al., 2014; Nagano et al., 2014; Kuriya et al.,
2015).
Local adaptation of floral traits to pollinators may have occurred
across multiple regions, but there is little evidence as to whether
variation in floral traits has occurred independently among regional
populations (but see Anderson et al., 2014; Toji et al., 2021). As in
textbook examples of ecological speciation (Nosil, 2012), one useful
approach to understand the interaction between trait diversification and
speciation in angiosperms is to combine a field analysis of local plant
evolutionary adaptations with a population genetic analysis that
examines genetic relationships among populations. In particular, local
adaptation of floral traits to pollinators may lead to speciation via
the establishment of prezygotic reproductive isolation (Grant-Stebbins
model; Grant & Grant; Stebbins, 1970; Johnson & Anderson, 2010;
Anderson et al., 2014), because one possible result of specialization of
a trait to a particular pollinator is a lack of pollinator sharing among
plant populations (Herrera et al., 2006; Anderson & Johnson, 2008;
Newman et al., 2015). About 25% of plant diversification events may be
associated with pollinator shifts (Van der Niet & Johnson, 2012); thus,
combined analyses of local adaptation of floral traits and population
genetics can shed light on the mechanisms of plant diversity (Thompson,
2005; Thompson et al., 2013). In this study, we conducted both trait and
genetic analyses to determine whether the differentiation of floral
traits (flower size) among plant populations in different mountain
regions was the result of secondary contact between two differentiated
lineages with long and short flowers, or whether flower size evolved
recently in each population as an adaptation to the local pollinator
size.
In genus Aquilegia (Ranunculaceae), adaptive radiation to
different pollinators (bumblebees, hummingbirds, and hawkmoths) has
occurred. Mainly, flower color, spur length, flower orientation, and
pistil length have evolved to adapt to each pollinator (Fulton &
Hodges, 1999; Hodges et al., 2004). Moreover, molecular phylogenetic
evidence also indicates that pollinator shifts have led to morphological
diversification and speciation within this genus (Whittall & Hodges,
2007). According to Whittall & Hodges (2007), a more ancestral floral
state of Aquilegia is purple, downward facing, short-spurred
flowers, which are pollinated by bumblebees. From plants with this
floral state, taxa with red, downward facing flowers with protruding
stamens and intermediate length spurs, which are pollinated by
hummingbirds, were derived. Then, taxa with white and yellow
long-spurred, lateral and upward facing flowers, which are pollinated by
hawkmoths, were derived from those taxa. Their results reveal an
interesting patten of species-level diversification as a consequence of
pollinator shifts, although evidence for flower trait diversification at
the earlier stages of speciation is lacking. To observe early stages of
speciation, it is useful to investigate the pattern of evolutionary
morphological diversification within a single species (Sobel &
Streisfeld, 2015).
In this study, we focused on evolutionary processes leading to spur
length and flower color differentiation in Aquilegia buergerianavar. buergeriana . In this species, geographic variation in spur
length has previously been observed in six populations in two mountain
regions, but the relationship between spur length and flower visitors in
these populations is not known (Hattori et al., 2014). Yellow-flowered
individuals are dominant in this species, and bumblebees seem to be the
main flower visitors. In some populations, red-flowered individuals
occur orthotopically with yellow-flowered individuals, but the genetic
relationship between red- and yellow-flowered individuals is unknown.
Differences in flower color in Aquilegia can lead to genetic
isolation even between neighboring or sympatric populations and is
likely to be important in speciation (Schemske & Bradshaw, 1999;
Hopkins & Rausher, 2012). Here, we first investigated the
correspondence between variation in floral spur length and
flower-visiting insect size in 16 Aquilegia populations in four
mountain regions. The results showed a morphological correlation between
spur length and average visitor size in each population, even within the
same mountain region; spur lengths were shorter in populations visited
by smaller flower visitors, and spur lengths were longer in populations
visited by larger flower visitors. Next, we identified genome-wide
single-nucleotide polymorphisms (SNPs) by the MIG-seq (multiplexed
inter-simple sequence repeat genotyping by sequencing) method (Suyama &
Matsuki, 2015) to clarify the genetic relationships among the
populations. These results showed that genetic relationships tended to
be clustered by mountain region and, therefore, that spur length evolved
in parallel in each mountain region. Individuals with different flower
colors were not differentiated in the genome-wide SNPs analysis,
however. This result suggests that pollinator isolation by flower color
has not occurred in these populations. Instead, the red flower color is
maintained in various populations in which most individuals have yellow
flowers.