Materials and Methods
To determine whether crossing success was impacted by floral similarity,
we quantified floral used in subsequent crossing trials. We genereated
data from five flowers per species for the following traits: length and
width of the corolla tube, throat and lobes, peduncle thickness, style
length, and ovary length. To determine whether crossing success was
influenced by vegetative similarity (vs. floral similarity, above) we
additionally quantified vegetative phenotypic divergence for these
species based on five leaves per species for the following traits: leaf
length, length width, petiole length, number of secondary veins, leaf
apex angle and leaf base angle. We used an Ocean Optics JAZ Spectrometer
to assess floral color differences following McCarthy et al. (2017).
Floral reflectance was measured three times per representative corolla
at a 45˚ angle. Resulting curves were averaged and then compared across
species. Overlapping spectra suggested five clear floral color bins
based on curve shape, reflectance wavelength, and median peak height:
purple, red, pink, yellow/green, or white (Supplementary Appendix).
To quantify the potential for hybridization, we attempted inter-specific
crosses for 16 species of Ruellia (Fig. 2) growing in controlled
environment glasshouses at University of Colorado. These species were
selected because they derived from the full geographical Neotropical
range of Ruellia , with some occurring regularly in sympatry and
others not. Because not all species flower at the same time, we were
able to attempt crosses between a total of 33 pairs of species, in both
directions. We focus on these pairwise comparisons when estimating
drivers of crossing success, including floral similarity and
geographical range overlap.
Hand pollinations. Hand pollinations were conducted on fresh,
fully anthetic flowers by brushing mature, pollen-coated anthers against
receptive stigmas (protocol adapted from Long 1966). This approach
mirrors the direct transfer of pollen by animal pollinators in natural
environments, which characterizes all species of Ruellia . Prior
to pollinations, pollen grains were assessed visually under 10x handlens
magnification for maturity, which is correlated to anther dehiscence inRuellia . To ensure pollen grain viability, one of the four
anthers produced by each species was removed and inspected using the
lactophenol-aniline blue stain protocol (Maneval 1936). Stigmas were
assumed to be receptive at the time of pollen maturity. For each cross,
we mimicked normal pollen load by estimating the average mass produced
by anthers of the maternal plant and then adjusting the dosage of pollen
donated by the paternal plant accordingly. All crosses consisted of
100% interspecific pollen. Pollinations were conducted between
09:00–17:00. Immediately following hand pollination, receptive flowers
were marked using a colored thread system to track multiple crosses on a
single individual. Threads were tied loosely but securely around floral
peduncles. A small pilot study conducted on flowers and leaves of six
species prior to implementation of the above tracking method indicated
that loose threading neither caused nor hastened tissue senescence over
a two-week period. Following visual inspection of seeds resulting from
successful crosses, one to several seeds per fruit were germinated to
further confirm cross success. We additionally attempted to germinate
seeds from crosses deemed to be unsuccessful based on visual assessment,
and none germinated.
All crosses were conducted carefully in a controlled environment in a
manner that emulates direct pollen transfer by animal pollinators.
Crosses were conducted reciprocally, alternating the donor/recipient
status in each cross (n=66 combinations in total for 33 species pairs).
The total number of attempted crosses for each combination varied from 2
to 50, with 88% of all species pair combinations being attempted at
least 10 times. Crosses were monitored daily until they were determined
to either fail or succeed. Crosses that failed to form fruits were
treated as failed crosses. Crosses that formed fruits but yielded
immature and/or non-viable seeds indicate embryo failure and were
treated as failed crosses. Fruits that yielded one or more mature,
viable seeds based on visual inspection followed by subsequent
germination trials were treated as successful crosses.
Molecular Methods. To account for potential effects of genetic
(i.e., phylogenetic) distances between species pairs, we employed the
matrix from Tripp and McDade (2014a), which was constructed using three
chloroplast markers plus the nuclear ITS+5.8S. We pruned this matrix to
contain only taxa relevant to the present study (Fig. 2). The new matrix
was aligned using PhyDE (Müller et al. 2016) then analyzed using maximum
likelihood imlemented in RAxML v8.2 (Stamatakis 2008). We then
constructed a temporally calibrated molecular phylogeny using BEAST
v1.82 (Drummond et al. 2012), with three fossil constraints
(Supplementary Table 1) derived from Tripp and McDade (2014b), to assess
temporal divergence between species pairs. Divergence time estimation
methods followed Tripp and McDade (2014b).
Statistical Analyses. To formally test for reproductive character
displacement in sympatric species pairs, we used a modified ANOSIM
(analysis of similarities) approach (Clark 1993). First, following Coyne
and Orr (1989) and Moyle et al. (2004), we classified a given species
pair as sympatric if the two species overlap in some portion of their
ranges. Co-occurrence was determined through collection notes and
localities of herbarium specimens and the extensive field data generated
by the first author and taxonomic expert on the genus. We quantified
overall reproductive character similarity as the mean Euclidean distance
between species in a multivariate decomposition of floral trait space,
derived from a principal component analysis of the correlation matrix of
the nine quantified floral characters. We also quantified the Euclidean
distance between species for each individual floral character. Measures
of Euclidean distance (or difference) between species for overall leaf
form and individual leaf characters were calculated in the same way.
Our modified ANOSIM approach consists of ranking in decreasing order the
Euclidean distances between all species pairs for a given character and
then calculating how different are sympatric species pairs, compared to
allopatric species pairs, for mean observed ranks. Specifically:
\begin{equation}
R_{\text{anosim}}=\ \frac{{\overset{\overline{}}{r}}_{s}-{\overset{\overline{}}{r}}_{a}}{\frac{n*(n-1)}{4}}\nonumber \\
\end{equation}Where \({\overset{\overline{}}{r}}_{s}\) equals the mean rank of
distances between sympatric species and\({\overset{\overline{}}{r}}_{a}\) equals the mean rank of distances
between allopatric species. The Ranosim statistic
varies from 1 to -1. Values of 0 would indicate that allopatric and
sympatric species pairs are no more different from each other than
expected by chance. A value of 1 would indicate that sympatric species
pairs are always more different in floral form for a given floral
character than allopatric species pairs, while a value of -1 would
indicate that allopatric species pairs are always more different. To
assess whether these differences between sympatric and allopatric
species pairs are significantly greater than expected by chance, we used
a permutation approach where we shuffled the rows and columns of the
dissimilarity matrix for a given character and obtained null
expectations for the R value, given the pairwise values being considered
(mimicking the same matrix permutation used in standard ANOSIM). This
controls for non-independence of data points involving the same species
when assessing significance. For a one-tailed test of the hypothesis
that sympatric species pairs will diverge significantly more for a given
trait than allopatric species pairs, we determined if the observed R
statistic was greater than that in 95% of the permutations.
To test if sympatric species pairs are more likely to differ in flower
color than allopatric species pairs, we conducted an initial chi-squared
analysis to assay whether these two categories of species pairs
(sympatric vs. allopatric) had different ratios of species pairs with
the same versus different flower colors. As this initial test showed no
difference (X2 = 0.01, p = 1), we did not pursue
additional analyses that would have controlled for non-independence of
data points.
In order to assess drivers of inter-specific crossing success, we used a
generalized linear mixed model (GLMM) framework to assess how
geographical range overlap and/or similarity in floral shape and color
and similarity in leaf shape impacted the success of interspecific
crosses. The response variable was the binomially distributed number of
successes and failures for each attempted cross. We included donor and
recipient species identities as random effects to control for
non-independence of crosses involving the same species and because both
donor and recipient species identities have significant effects on
crossing success (likelihood ratio tests of binomial GLM with species
identify as fixed effect versus null model, for donor:
Χ2 = 71.2, p < 0.001; and recipient:
Χ2 = 95.2, p < 0.001). There was no
relationship between crossing success in one direction versus the other
(Supplementary Fig. 1; r = 0.06, p = 0.751), and including individual
species pairs as a random effect did not improve our statistical models
or change estimates of fixed effects. We also included the genetic
distance between species as a fixed effect in analyses to control for
this additional potential driver of crossing success. We first compared
the performance of models with a single fixed effect (and the random
effects) to models with only random effects using likelihood ratio
tests. We then constructed a full model with all fixed and random
effects and compared this full model to sub-models where each fixed
effect was dropped in turn, again using likelihood ratio tests. The
formula for the full model is:
bin(number of crosses, probability of success) ~ flower
colour similarity + floral shape similarity + leaf shape similarity +
genetic distance + allopatry vs. sympatry + (1 | Recipient
Species Identity) + (1 | Donor Species Identity)
We tested for model overdispersion using a chi-squared test with the
residual deviance and degrees of freedom. We did not attempt to test for
interactions between our fixed effects due to limited sample size.
While our statistical approach accounts for non-independence of data
points due to the same species being used in multiple crosses and to
variation in phylogenetic relatedness of species (following Tobias et
al. 2014), and while also correctly modeling our binomially distributed
crossing success data, it is not identical to ‘phylogenetically
corrected’ approaches used in previous studies that tested the effect of
sympatry vs. allopatry on reproductive isolation. In order to ensure
comparability with previous studies, we conducted an additional
statistical test following procedures used by Coyne and Orr (1989) and
Moyle et al. (2004). Specifically, we averaged the proportion of
successful crosses for all pairs of species that span a given node in
our phylogeny to yield a single estimate of crossing success for each
node in the phylogeny. Four of the nodes in our phylogeny were not
spanned by any species pair in our study and were omitted from further
analysis. Seven nodes in the phylogeny have only allopatric species
pairs spanning them, while four nodes have sympatric species spanning
them. We compared the mean crossing success values for nodes with only
allopatric species pairs to that for nodes spanned by sympatric species
pairs using a one-tailed non-parametric Wilcoxon test.