Discussion
The contribution of RCD to Floral Diversity. In plants, a pattern
of increased floral divergence among sympatric compared to allopatric
populations of species is well-documented (Whalen 1978; Armbruster et
al. 1994; Fishman and Wyatt 1999; Coyne and Orr 2004; Muchhala and Potts
2007; Norton et al. 2015; Gögler et al. 2015; Koski and Ashman 2016;
Lagomarsino and Muchhala 2019). In this study, we document clear
patterns of reproductive character displacement (RCD) in sympatric
species of a diverse lineage of Neotropical plants. This adds to a
growing body of evidence for pervasiveness of the pattern in plant
communities (Grossenbacher and Whittall 2014; Grossenbacher and Stanton
2014; Norton et al. 2015; Koski and Ashman 2016), extending significance
of the phenomenon documented in tropical flowering plants. Our data in
tandem with results from numerous prior studies suggests that RCD may be
a major factor underlying the great floral diversity of angiosperms. The
import of RCD is likely intensified among tropical latitudes given the
density and diversity of plant-pollinator interactions.
Evidence in Support of Reinforcement . Two principle mechanisms
help explain patterns of RCD: pollinator competition (Muchhala et al.
2014) and reinforcement (Kay and Schemske 2008; Hopkins 2013). Character
displacement of floral traits can, specifically, arise from competition
for pollinators in sympatry between species that are already
reproductively isolated (Muchhala et al. 2014). However, our assessment
of which flower characters diverge more between sympatric species pairs
than allopatric species pairs combined with results from our artificial
crossing experiments suggest a role for reinforcement in Ruellia .
Using hand pollinations in a controlled glasshouse environment, we found
exceptionally high prezygotic isolation between sympatric species pairs:
only one of 95 crossing attempts across five sympatric species pairs
produced any viable seeds. In contrast, crosses between allopatric
species pairs (n = 28 pairs) were 8x more likely to be successful (9%
success rate over all crosses; 30% of crossed allopatric species pairs
were successful at least once). We assessed how similar crossed species
pairs were in the shape and color of their flowers, and our results
suggest that divergence in floral shape, especially style length, may be
one underlying mechanism that results in strong barriers to reproductive
compatibility between sympatric species. Both results – strong
reduction in crossability between sympatric compared to allopatric
species pairs and divergence in morphological features not likely under
selection by pollinators – better fit a model of reinforcement over
pollinator competition. In this study, we also found that species with
differently colored flowers also have greatly reduced crossing success,
but sympatric species pairs were no more likely to have similar or
differently colored flowers than allopatric species pairs. It may be
that reinforcing selection acts more strongly on style length than it
does on flower color.
Pollinators likely select flowers based on their overall reward, shape,
and color. In contrast, it is less likely that they select flowers based
primarily on style length. Yet, we have shown that style length diverges
more between sympatric species pairs relative to allopatric species
pairs than any other floral trait measured (Fig. 3). All sympatric
species differ in style length by at least 21.8 mm, while 23 of 28
allopatric species pairs differ in style length by lesser amounts (Fig.
3). If competition for pollinators, absent any involvement of
reinforcing processes, were driving patterns recovered in our dataset,
we would expect all or most floral traits to show divergence in sympatry
instead of the highly variable divergence among characters we recovered
(Fig. 3). Further, style length is a key character that may underlie
potential incompatibilities (Kay and Schemske 2008), and selection on
style length would serve to generate a pre-zygotic barrier between
species where hybridization is maladaptive. Nonetheless, we recognize
that reinforcing selection and pollinator competition are not always
mutually exclusive: severe costs of pollen transfer between related
species in sympatry may involve both types of processes when
reproductive isolation is not fully complete and species boundaries
remain semipermeable (Grossenbacher and Stanton 2014; Harrison and
Larson 2014; Muchhala et al. 2014).
Two important criteria for reinforcing selection to occur are (i) that
hybridization between species pairs is possible such that it can be the
subject of selection, and (ii) that hybridization is maladaptive and
selected against (Kay and Schemske 2008; Hopkins 2013). We have shown
that crosses between 30% of allopatric species pairs are capable of
producing viable seeds. Thus, hybridization is at least possible, as was
earlier demonstrated by Long (1975). We do not have experimental
evidence for the second criterion, but we note that hybrids inRuellia are rare in natural environments despite extensive
geographical range overlap and contemporaneous flowering periods among
numerous sets of species, suggesting it is maladaptive to be a hybrid
(or that hybridization is otherwise rare to begin with). The first
author (E. Tripp) has seen and studied nearly 200 of 300 Neotropical
species in their native habitats (www.trippreport.com/ruellia-pages) and
in only one instance has a natural hybrid been encountered (i.e.,
between Ruellia brevifolia and R. puri ; Bolivia, E.
Tripp et al. 5971 & 5977 [COLO Herbarium]; only three
putative, additional natural hybrids have been reported by other authors
in the literature: Daniel 1990 [Ruellia amoena & R.
foetida ]; Ezcurra 1993 [Ruellia brevifolia & R.
longipedunculata ; R. brevicaulis & R. coerulea ]).
Finally, increased floral divergence among sympatric compared to
allopatric populations of species is exceptionally well-documented in
plants, and reinforcement against maladapted hybrids is likely to
account for at least some of these instances. That reinforcing selection
may sometimes involve action on only a single locus (Hopkins and Rausher
2012) suggests the potential for relatively simplistic genetic framework
underlying contributions of reinforcement to the evolution of RI. Taken
together, both the potential frequency and simplicity underlying
reinforcement suggest that this type of selection may commonly
contribute to completing the process of RI (Liou and Price 1994).
However, a paucity of studies that have employed comparative data across
clades to investigate RCD and its drivers has precluded understanding of
whether such processes help drive lineage diversification and, if so,
just how pervasive these processes are (Servedio 2004; Yukilevich 2012;
Hopkins 2013). That sympatric species pairs in Ruellia are more
reproductively isolated and more divergent in floral morphology than are
allopatric pairs provides evidence in support of reinforcement.
Alternatively, if reproductive barriers between species in a given
lineage are incomplete, sympatric species that are similar in floral
morphology may interbreed due to sharing of pollinators coupled with
mechanical and genetic compatibility (Templeton 1981). If gene flow
between these diverged yet reproductively compatible lineages is
recurrent and prolonged, such lineages may ‘fuse’, likely with the more
fit or otherwise more abundant species in a given environment
genetically swamping the less fit, less abundant species (Webb et al.
2011). Meanwhile, sympatric species that are highly dissimilar in floral
form may be unable to interbreed and maintain distinct evolutionary
lineages boundaries. Thus, ‘differential fusion’ (Templeton 1981) can
yield a pattern of RCD similar to that driven by reinforcement. Although
we cannot fully rule out differential fusion in this study, under such a
model (and in contrast to reinforcement), natural hybrids should be
commonly observed in nature between incompletely isolated lineages.
However, as discussed above, natural hybrids are exceedingly rare inRuellia . Additionally, extensive evidence now documents
reinforcement even in the face of gene flow (Matute 2010; Roda et al.
2017).
Additional Drivers of Variation in Crossing Success . Consistent
with studies of model systems in animal speciation biology (Coyne and
Orr 2001), we found that crossing success declines with increasing time
of evolutionary divergence between species pairs. Whereas it is well
established in animals that genetic distance is a significant predictor
of interspecific fertility (Coyne and Orr 1989), there has historically
been less consensus in plants (Edmands 2002; Moyle et al. 2004). Moyle
and colleagues (2004) used comparative data from multiple species to
demonstrate that increasing genetic distance strongly decreased
crossability in one of the investigated study systems (Silene ),
but not in the other two lineages they examined. Similarly, using a
massive dataset on species crossability in Eucalyptus , Larcombe
et al. (2015) found decreased reproductive compatibility with increasing
genetic distance. Our and other studies (e.g., Scopece et al. 2007;
Moyle et al. 2014; Brandvain et al. 2014) confirm a growing generality
of this pattern in plants. The implications of this generality with
respect to reinforcement are that reinforcement, as a process important
to speciation, should scale with genetic distance. That is, at shallower
levels of evolutionary divergence, reinforcement may potentially play a
greater role in RI than it does at comparatively deeper evolutionary
divergences. This prediction should be tested in a carefully controlled
experiment focused on a series of species pairs that vary in their
degree of phylogenetic relatedness but otherwise have in common other
life history attributes.
Variation in Reproductive Character Displacement Across Clades and
Latitudes . In addition to genetic distance, at least four factors
should increase opportunities for and thus the potential impacts of both
reinforcing selection and pollinator competition in driving floral
divergence in sympatry: clade taxon richness, geographically
wide-ranging and overlapping species, high densities of individuals
within populations, and consequences of gene flow. In Ruellia ,
hundreds of species span one of the largest latitudinal gradients
occupied by any lineage of flowering plants: ca. 80 degrees (i.e., from
~43˚N near Milwaukee, Wisconsin to ~37˚S
in central Argentina). Over half of the ~300 New World
species have broad geographical ranges (i.e., ranges that extend beyond
the borders of a single country). Additionally, there exists widespread
co-occurrence of both closely related and more distantly related species
in Ruellia , and populations often consist of tens to hundreds of
individuals (Tripp 2007; McDade and Tripp 2007; Tripp 2010; Tripp and
Luján 2017). Species-rich lineages in which close relatives commonly
encounter one another in natural environments should, on the whole,
witness greater opportunity for floral diversification via either
reinforcing selection or pollinator competition. If these opportunities
involve maladaptive gene flow, then reinforcement is expected to be
strong and fast acting to reduce energetic costs of producing unfit
hybrids. If the above predictors are accurate, emergent properties
associated with lineages such as total species number, degree of range
overlap, and phylogenetic relatedness of co-occurring species should
help predict the relative frequency and importance of underlying drivers
of reproductive character displacement in natural landscapes.
We expect that these emergent characteristics of lineages associated
with opportunity for reinforcing selection and/or pollinator competition
should be more pronounced in tropical (compared to temperate) latitudes,
where there typically exists much greater taxonomic and functional
diversity of pollinators. Thus, variation in phenomena such as
reinforcement across latitudes may be one mechanism contributing to
latitudinal gradients in sympatric, and perhaps overall, biodiversity.Ruellia and other broadly ranging lineages (e.g.,Asclepias ) provide excellent systems in which to study whether
and how processes including reinforcement and, presumably, competition
vary with latitude in plants.