Introduction
“Endless forms most beautiful” have motivated biologists for centuries
(Darwin 1859; Carol 2005), and the remarkable floral diversity of
angiosperms is one prime example. Floral diversification in many
angiosperm clades is linked to interactions with animal pollinators,
given that most angiosperms (~88%) are animal
pollinated—a number that rises to 94% within tropical plant
communities (Ollerton et al. 2011). Pollinators have behavioral
preferences for different rewards, forms, and colors of flowers, which
has contributed to a remarkable range of floral diversity (Sargent 2004;
Waser and Ollerton 2006; Chittka and Raine 2006; Tripp and Manos 2008;
Johnson 2010; Dudash et al. 2011; Van der Niet & Johnson 2012; Gervasi
and Schiestl 2017; Smith and Kriebel 2017). When close relatives within
a lineage occur in sympatry and are adapted to similar functional groups
of pollinators, pollinator competition can arise and negatively impact
fitness of one or both plant species (Caruso 2000; Grossenbacher and
Stanton 2014; Muchhala et al. 2014; Sletvold et al. 2016). In such
instances, selection for floral divergence in sympatry can arise, which
has been documented in numerous groups of flowering plants, especially
in temperate angiosperms (Sletvold et al. 2016). Pollinator competition
can thus lead to greater floral divergence in sympatry compared to
allopatry, or reproductive character displacement (RCD; Grossenbacher
and Stanton 2014), which represents an important mode of ecological
character displacement sensu the classical definition (MacArthur
and Levins 1967).
Other mechanisms in addition to pollinator competition can lead to
reproductive character displacement, although only some can be
attributed to direct selection for character displacement. One such
mechanism—reinforcement—results from direct selection to reduce gene
flow between sympatric, diverging (or already divergent) lineages
(Wallace 1889; Coyne and Orr 1989; Matute 2010; Hudson and Price 2014;
Hopkins and Rausher 2012). Reinforcement, which has been documented in a
limited number of plant lineages, these primarily in temperate regions,
describes the process whereby previously allopatric lineages experience
selection to avoid costly hybridization after coming into sympatry. In
angiosperms, reinforcing selection often operates on floral morphology,
thus driving the evolution of morphological divergence in floral traits
(Grant 1966; Moyle et al. 2004; Silvertown et al. 2005; Kay and Schemske
2008; Hopkins and Rausher 2012). The underlying assumption of
reinforcement is that hybridization is costly because it fails to yield
offspring, or offspring have reduced fitness compared to non-hybrid
offspring. Reinforcement is often thought to ‘complete’ the speciation
process that begins when populations of species become isolated in
allopatry but then later come into contact. While many classic studies
of Drosphila and other animals support the concept of
reinforcement, it has remained more controversial and less
well-documented in plant evolutionary biology (reviewed in Hopkins
2013). Reinforcing selection, if common, is thought to act quickly such
that natural hybrids are rarely observed.
Distinguishing between pollinator competition and reinforcement as
primary drivers for RCD remains difficult despite the importance of
understanding mechanisms that drive plant species divergence and floral
diversification. In this study, we propose a two-step approach to help
distinguish between these two processes, and then apply this approach to
understand floral divergence in sympatry in a species-rich lineage of
Neotropical angiosperms (Ruellia L.: Wild Petunias; Fig. 1). The
first step involves emphasis on the floral characters themselves that
underlie RCD. Pollinators typically select flowers based on visual and
olfactory cues that signal reward (nectar and pollen, primarily) and
thus divergence in these and associated characters, i.e., color, tube
length, and tube width, which frequently co-vary with reward, may signal
pollinator competition (Ornelas et al. 2007; Benetiz-Vieyra et al. 2014;
Knauer and Schiestl 2014). In contrast, under reinforcement, selection
may include traits related to pollinator preference, as above, but is
likely to involve additional mechanical forms of isolation or structural
incompatibilities that prevent cross fertilization (Kay and Schemske
2008; Hopkins 2013). Thus, divergence in other traits not typically
associated with pollinator preference, such as style length or pollen
tube length, lends support to hypotheses of reinforcement over
pollinator competition.
As a second step, artificial cross pollinations and resultant data on
reproductive incompatibility (RI) can be employed to help further
distinguish pollinator competition from reinforcement. Under pollinator
competition alone as the primary driver for RCD, selection should act to
reduce visitation of a given pollinator to different plant lineages
(species or incipient species), but other mechanisms to prevent
hybridization such as mechanical or intrinsic isolating factors are not
expected to manifest between plant lineages. In contrast, under
reinforcement, plant lineages divergent in floral morphology should be
recalcitrant to artificial hybridization because of mechanical
incompatibilities that arise to prevent further, maladaptive
hybridization. Hand pollinations bypass the action of pollinators and
therefore offer additional means to distinguish between reinforcement
and competition for pollinators. If hand pollinations between lineages
with dissimilar flowers consistently yield non-viable offspring,
reinforcement may be a primary driver of RCD. In contrast, if such hand
pollinations do consistently yield viable offspring, then pollinator
competition may instead be a primary driver of RCD.
In this study, we examine a species-rich and florally diverse lineage of
tropical angiosperms to (1) test for RCD between species pairs and then
(2) evaluate evidence in support of two different mechanisms that
contribute to RCD: pollinator competition and reinforcement. We first
determine which characters show the strongest pattern of RCD between
species pairs. Second, we use hand pollinations in a carefully
controlled glasshouse environment to test whether floral dissimilarity
is correlated with RI. Finally, we assess if species pairs show greater
post-pollination RI when in sympatry compared to allopatry by
incorporating geographical range overlap as well as other potential
effects, specifically phylogenetic relatedness. Finding that
dissimilarity in floral traits (that are unlikely selected for by
pollinators) is correlated with RI and that sympatric species cannot
produce viable offspring is here taken as evidence in support of
reinforcement, whereas finding that floral dissimilarity is unrelated to
RI is taken as evidence in support of pollinator competition. The
results from this study have implications for understanding the relative
contribution of RCD to floral diversification, especially given few
examples are known from the tropics (but see Kay and Schemske 2008;
Muchhala et al. 2014), and serve as steps towards disentangling the
underlying drivers of RCD.