Int. J. Plant Sci. 181(7):000–000. 2020.

q 2020 by The University of Chicago. All rights reserved.

1058-5893/2020/18107-00XX$15.00 DOI: 10.1086/708906

EVOLUTION OF FLORAL MORPHOLOGY AND SYMMETRY IN THE MICONIEAE

(MELASTOMATACEAE): MULTIPLE GENERALIZATION TRENDS
WITHIN A SPECIALIZED FAMILY
Maria Gavrutenko,1,* Marcelo Reginato,†,‡ Ricardo Kriebel,§ Antoine N. Nicolas,∥ and Fabián A. Michelangeli†
*Department of Biology, City College of New York, City University of New York, 160 Convent Avenue, New York, New York 10031, USA; †Institute
of Systematic Botany, New York Botanical Garden, 2900 Southern Boulevard, Bronx, New York 10458, USA; ‡Departamento de Botânica,
Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 90650-001, Brazil; §Department of Botany,
University of Wisconsin–Madison, Madison, Wisconsin 53706, USA; and ∥Manhattan College,
4513 Manhattan College Parkway, Riverdale, New York 10471, USA

Editor: Gerhard Prenner

Premise of research. Analyses of the evolution of floral morphology and symmetry broaden our understanding
of the drivers of angiosperm diversification. Integrated within a flower, labile floral characters produce different
phenotypes that promote variable interactions with pollinators. Thus, investigation of floral evolution may help
with inferring potential historic transitions in pollinator modes and ecological pressures that generated present di-
versity. This study aims to explore the morphological evolution of flowers in Miconieae, a species-rich Neotropical
tribe within the Melastomataceae. Despite a constrained floral plan, Melastomataceae manage to achieve a variety
of floral phenotypes appealing to diverse pollinator types, with a majority of the species requiring specialized “buzz
pollination” by bees. However, previous research of Miconieae documented several instances of convergent evolu-
tion of phenotypes associated with generalized pollination strategies. We explore the floral morphological evolution
of Miconieae in a phylogenetic context to understand how diversification relates to different phenotypes and how
common evolution of generalized pollination systems is within this tribe.
Methodology. We constructed the largest species-level phylogeny of the Miconieae and combined it with mor-
phological data on a variety of floral characteristics for more than 350 species scored from field photographs. Trait
evolution was then analyzed using ancestral state reconstruction with stochastic character mapping. Trait corre-
lation was estimated with Pagel’s statistical method for discrete characters and with regression analysis of phylo-
genetically independent contrasts of continuous characters. We analyzed diversification in the tribe with Bayesian
analysis of macroevolutionary mixtures and explored the effect of character state evolution on diversification with
the binary state speciation and extinction approach.
Pivotal results. Our analyses reveal rampant convergent and correlated evolution of multiple characters in-
dicative of pollinator-mediated selective pressures.
Conclusions. We confirm several parallel trends in the evolution of generalized floral phenotypes and find an
association between generalization trends and increased diversification rates that may be related to colonization
of highland environments.

Keywords: generalization, specialization, pollination syndromes, diversification rates, herkogamy, floral traits.

Online enhancements: supplemental tables, figure, data. Dryad data: https://doi.org/10.5061/dryad.c2fqz614s.

Introduction many evolutionary and taxonomic studies aimed at explaining

The flower is a complex reproductive structure and an inte-
grated system of constituent floral parts (Faegri and van der Pijl
1979). Floral morphology has understandably been a focus of
1
Author for correspondence; email: gavrucent@gmail.com.
Manuscript received August 2019; revised manuscript received January 2020;
electronically published August 6, 2020.
the observed diversity of angiosperms (Anderson et al. 2002).
Floral parts carry out distinct functions with a goal of successful
sexual reproduction, which makes the entire system subject to
evolutionary forces. Exploring variability in floral morphology
in a phylogenetic framework may point to drivers of evolution
within a clade.
Floral morphology can be interpreted as the result of concerted
adaptation of multiple floral traits to selective pressures exerted by
pollinators in interactions that vary in their degree of specialization;

000

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000 INTERNATIONAL JOURNAL OF PLANT SCIENCES
this is the idea at the heart of the pollination syndrome theory
(Fenster et al. 2004; Rosas-Guerrero et al. 2014). Classical polli-
nation syndromes in functionally specialized interactions involve
phenotypic adaptations of multiple floral traits to attract and fit a
certain pollinator type and are well documented (Faegri and van
der Pijl 1979; Waser et al. 1996; Fenster et al. 2004). On the other
hand, generalist flowers are not strongly adapted to a certain
functional guild of pollinators and thus may not display easily
classified phenotypes, a complexity that perhaps reduces the ap-
peal of these systems for researchers (Ollerton et al. 2007). Further
complexity is added by reports of intermediate pollination sys-
tems with more than one specialized effective pollinator or vary-
ing degrees of generalization observed in species transitioning
from one specialized pollinator to another (Manning and Gold-
blatt 2005; Rosas-Guerrero et al. 2014; Dellinger et al. 2019b).
The existence of the continuum of plant-pollinator interactions
from generalized to specialized means that not all flowers neatly
conform to idealistic syndromes, complicating the inference of
agents of evolution from floral morphology (Ollerton et al.
2009). However, a concurrent examination of the evolution
of several floral traits through the prism of pollinator inter-
actions that they promote may provide the context for clade di-
versification, specifically the evaluation of transitions between
specialization and generalization. Such an investigation is par-
ticularly intriguing within Melastomataceae, a plant family as-
sociated with a wide variety of generalist and specialist pollina-
tors despite its relatively conserved flower morphology (Renner
1989; Brito et al. 2016).
Melastomataceae is a diverse family with more than 4500 spe-
cies (Renner 1993; Michelangeli et al. 2004). Melastomataceae
manage to achieve a variety of floral traits necessary to appeal
to diverse pollinator types, despite a constrained floral plan (com-
plete flowers with free floral parts and no fusion within or between
whorls). The majority of Melastomataceae offer pollen as a re-
ward and require specialized “buzz pollination” by bees (Renner
1989), a pollinating strategy that convergently evolved across
multiple angiosperm orders (De Luca and Vallejo-Marín 2013).
In these specialized flowers, pollen is enclosed in tubular anthers
with usually minute terminal pores, and it is released only when a
bee grasps a stamen and vibrates the anther at a high frequency
using its flight muscles (Buchmann 1983; Renner 1989; Vallejo-
Marín 2019). Divergences from buzz pollination to other modes
are widespread and often involve a transition to other specialized
pollination systems, often with vertebrate pollinators (Pérez et al.
2006; Martén-Rodríguez et al. 2010; Rosas-Guerrero et al. 2014;
van der Niet et al. 2014). Indeed, other specialized modes of pol-
lination have evolved several times within the Melastomataceae,
with some flowers providing nectar rewards to hummingbird, ro-
dent, and bat pollinators (Lumer 1980; Renner 1989; Judd 2007;
Dellinger et al. 2019a, 2019b) or food body rewards to passerines
(Dellinger et al. 2014, 2019a). Transitions from highly specialized
and effective bee pollination to generalist insect pollinator assem-
blages are less frequent and need further study (Rosas-Guerrero
et al. 2014). In Melastomataceae, independent evolution of gen-
eralized pollination systems involving flies, wasps, butterflies,
and nonbuzzing bees has been reported in several instances
(Goldenberg 2008; Kriebel and Zumbado 2014; Brito et al.
2016, 2017).
Shifts in pollination mode have been suggested to be evolu-
tionary drivers of variability in floral morphology in Melastoma-
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taceae and specifically of stamen diversification in Miconieae
(Goldenberg et al. 2008; Luo et al. 2009; Brito et al. 2016;
Dellinger et al. 2019a). Miconieae is a wide-ranging, phylogenet-
ically complex Neotropical tribe of approximately 1800 species
(19–23 genera; Michelangeli et al. 2008). Variation in stamen
morphology within Miconieae is correlated with shifts to gener-
alized pollination systems: short broad-pored anther phenotypes
visited by multiple pollinator groups evolved several times in the
tribe, where a majority of species display minute-pored longer
anthers requiring vibrating action by buzzing bees for pollen re-
lease (Brito et al. 2016). Interestingly, parallel evolutionary trends
in floral morphology seem to have occurred multiple times, sug-
gesting that ecological pressures have led to the convergent evo-
lution of similar phenotypes (Goldenberg et al. 2008; Reginato
and Michelangeli 2016). Moreover, previous research detected
the correlated evolution of floral structures, anther color and in-
florescence architecture, and existence of distinct morphological
regimes within one diverse lineage of Miconieae, potentially in-
dicative of pollinator transitions among different groups of
buzzing bees (Reginato and Michelangeli 2016). Shifts toward
more generalized pollination strategies within the tribe have of-
ten been interpreted as a means to increase reproductive success
in depauperate environments devoid of bees, such as the high-
lands (Cruden 1972; Sandvik et al. 1999; Goldenberg et al.
2008; Varassin et al. 2008; Brito et al. 2017). As a species-rich,
morphologically diverse, and widespread tribe, Miconieae pres-
ents a great study case for investigating the ubiquity of transi-
tions toward generalized pollination in a phylogenetic context,
as such studies are few and have often been carried out on island
systems (Armbruster and Baldwin 1998; Kay and Sargent 2009;
Martén-Rodríguez et al. 2010).
In this exploratory study of morphological evolution within
Miconieae, we combine an extensive morphological data set
with a species-level phylogeny to understand how diversifica-
tion within this tribe relates to floral form. Floral morphology
and symmetry and the variable pollinator interactions promoted
by different floral phenotypes have repeatedly been implicated in
diversification rate variability in angiosperms (Givnish 2010;
O’Meara et al. 2016; Vamosi et al. 2018). Here, we dissect the
conserved floral plan into multiple constituent traits and analyze
the evolutionary history of the individual floral characters to ex-
amine the origins of phenotypic diversity in a seemingly evolu-
tionarily constrained tribe. We relate these labile floral charac-
ters to their function in pollinator attraction and orientation
to consider where such morphological adaptations fall on the
specialization-generalization continuum. We also examine traits
that characterize the entire flower, such as symmetry of repro-
ductive whorls (androecium and gynoecium) and herkogamy
(spatial separation of pollen and stigma to promote outcrossing).
Finally, we analyze trait correlations and diversification rates
within the tribe to answer the following questions: (1) How is di-
versification in the tribe related to different suites of morpholog-
ical features? (2) How do different character states of floral traits
contribute to phenotypes on a continuum from specialization to
generalization? (3) How common is the evolution of generalized
pollination systems in Miconieae? We expect integrated evolu-
tion of floral traits and multiple instances of convergent evolu-
tion of trait combinations indicative of generalization and antic-
ipate gaining insights for further targeted studies within this
speciose group.
 GAVRUTENKO ET AL.—MORPHOLOGY AND SYMMETRY IN MICONIEAE FLOWERS
Methods
Molecular Data Set and Phylogeny
To study the evolution of floral morphology and symmetry,
we generated the most densely sampled phylogeny of the Mico-
nieae to date. The molecular data set (5584 sequences) comprised
sequences available in GenBank from previously published stud-
ies (Michelangeli et al. 2004, 2008; Goldenberg et al. 2008; Mar-
tin et al. 2008; Reginato et al. 2010; Gamba and Almeda 2014;
Kriebel et al. 2015; Majure et al. 2015a, 2015b; Meirelles et al.
2015; Reginato and Michelangeli 2016), sequences available in
GenBank but not used to date in any published studies, and
1025 sequences (18.4% of all sequences) newly generated for this
work (app. 1; apps. 1, 2A, 2B are available online). The molecu-
lar matrix included three nuclear markers (waxy, nrITS, and
nrETS) and six plastid markers (trnS, psbK, accD, atpF, ndhF,
and rbcL). The Miconieae were represented by 1083 species from
17 genera. The outgroup included 81 species from the Meria-
nieae (six genera), one species of Eriocnema, and three species
of Physeterostemon (see Clausing and Renner 2001; Michel-
angeli et al. 2004, 2008; Goldenberg and Amorim 2006; Golden-
berg et al. 2008).
DNA from previously unsampled species was extracted from
silica-dried leaf tissues using the Qiagen DNeasy Plant Mini Kit
(Qiagen, Valencia, CA) following the manufacturer’s protocol
with the modifications outlined by Martin et al. (2008). Four
DNA regions were amplified with polymerase chain reaction fol-
lowing protocols described in Michelangeli et al. (2004; nrITS1–
nrITS2), Shaw et al. (2005; accD), Reginato et al. (2010; psbKL),
and Kriebel et al. (2015; nrETS). Sequences were aligned using
MAFFT version 7 (Katoh and Stanley 2013) through a plug-in
for Geneious 9.0 (http://www.geneious.com; Kearse et al. 2012).
Phylogenetic relationships among species were reconstructed by
maximum likelihood using RAxML-HPC BlackBox through the
CIPRES portal (http://phylo.org; Miller et al. 2010) on the basis
of a nine-marker alignment (10,851 bp). Divergence times were
estimated with a penalized likelihood approach for large phy-
logenies (Sanderson 2002) implemented in treePL (Smith and
O’Meara 2012). We provided nine secondary calibration points
(table S1; tables S1–S3 are available online). These age estimates
were extracted from a Melastomataceae phylogeny (Caetano
et al. 2018), and we consider them to be more accurate than the
previously estimated date of Miconieae origin (27.1 mya; Berger
et al. 2016), which included only a few species of the group. The
chronogram from treePL was used for all downstream analyses
of diversification and ancestral state reconstructions.
Morphological Data Set
To study the evolution of floral morphological traits, we
scored 358 species of Miconieae. Morphological characters were
scored from field photographs of flowers at anthesis. Images are
available at http://sweetgum.nybg.org/melastomataceae, http://melas
-centroamerica.com, and in the Dryad Digital Repository (https://
doi.org/10.5061/dryad.c2fqz614s; Gavrutenko et al. 2020). A
morphological matrix was built in Xper3 (http://xper3.fr) and in-
cluded discrete and continuous morphological characters. We
defined 12 core characters describing floral form as well as the
symmetry and morphology of reproductive organs (app. 2A),
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000
expected to be under strong selection because of their role in flo-
ral function and pollinator interactions (Kay and Sargent 2009;
O’Meara et al. 2016). However, because of the constraints im-
posed by the conserved floral plan of Miconieae, these characters
did not fully capture the various aspects of morphological dispar-
ity in the tribe. To further characterize phenotypic variability,
we defined 11 additional exploratory characters (app. 2B; sup-
plementary material VII). Some characters were nested and in-
applicable to some species in the phylogeny and thus did not par-
ticipate in the downstream analyses. However, they served to
augment our understanding of the suite of core characters and
to present a more complete view of Miconieae-specific aspects
of variability. The full list of character descriptions and exam-
ples is presented in the supplementary material.
Ancestral State Reconstruction
To analyze discrete trait evolution, we performed ancestral
state reconstructions using stochastic character mapping (Boll-
back 2006) with the function make.simmap of the R package
phytools version 0.5-64 (Revell 2012). We pruned the chrono-
gram obtained from treePL analyses to remove the species not
sampled in the morphological data set. We first estimated the
optimal model of character evolution with the fitDiscrete func-
tion of the R package GEIGER version 2.0.6 (equal rates model
for style direction; all rates different [ARD] model for all other
discrete characters; Harmon et al. 2008) and then conducted
1000 simmap simulations with an estimated root prior. For con-
tinuous characters, we log transformed the data and estimated
ancestral states with the function contMap from the package
phytools (Brownian motion model; Revell 2012). Character
states for all examined traits were plotted on the dated phylog-
eny to visualize patterns in the data.
Trait Correlation
Correlations between discrete binary characters were ana-
lyzed with Pagel’s statistical method (Pagel 1994) implemented
in phytools (Revell 2012) with Bonferroni correction for multi-
ple pairwise comparisons. This method fits two models to the
data: a model in which traits evolve independently of each other
and a model in which the evolution of two characters is corre-
lated. A likelihood ratio test is then used to assess whether the
difference between models is significant. Although this test was
designed to correct for phylogenetic pseudoreplication, this
method has been criticized in the literature because it often infers
statistically significant correlations, even when the similarity in
traits is an attribute of common evolutionary origin (Maddison
and FitzJohn 2015). However, the large size of the Miconieae
phylogeny and the ample variation present in the traits studied
here suggest that pseudoreplication is not a problem. Tests were
carried out using the fitPagel function with the fitMk method for
the optimization and the ARD evolutionary model for each char-
acter. Two nonbinary characters (anther symmetry and pore size)
were constrained to only two states per trait to fit test require-
ments. We converted anther symmetry to a binary character by
grouping monosymmetric and asymmetric anthers into one cat-
egory because, as opposed to polysymmetric anthers, both char-
acter states may be indicative of specialized pollination. Pore size
was reclassified into minute pores versus large pores by merging
000 INTERNATIONAL JOURNAL OF PLANT SCIENCES
broad pores with rimose pores because neither of these character
states requires specialized buzz pollination.
Correlation between continuous traits was evaluated with
regression analysis of phylogenetically independent contrasts.
Phylogenetically independent contrasts were calculated follow-
ing the comparative method introduced by Felsenstein (1985)
to account for nonindependence of individual species within a
phylogeny. The analysis was performed using the pic function in
the R package APE (Paradis et al. 2004).
Diversification Analyses
We analyzed diversification in the tribe with Bayesian analysis of
macroevolutionary mixtures (BAMM; Rabosky 2014). BAMM
models diversification rates with the reversible-jump Markov
chain Monte Carlo approach and is designed to detect rate hetero-
geneity and shifts in evolutionary regimes within the system. We
assigned sampling probability to clades on the basis of the estimate
of sampled diversity of each clade using previously published
monographs of the tribe and our own expert taxonomic knowl-
edge (Renner 1993; Michelangeli et al. 2004, 2008; Goldenberg
et al. 2008; Martin et al. 2008; Reginato et al. 2010; Kriebel
2016; Reginato 2016). We retained one outgroup clade, Eriocnema
and Physeterostemon, because it was well sampled. Analyses were
run for 80 million generations, sampling every 7000 generations,
with the first 10% of samples discarded as burn-in. To address po-
tential sensitivity of the results to the prior on the expected num-
ber of shifts (Moore et al. 2016), we tested several priors and, as
the analyses were robust to prior choice (fig. S1, available online),
set the prior on expected number of shifts to 10 (as suggested
by BAMMtools for a phylogeny of this size; Rabosky et al.
2014). The best rate shift configuration was obtained with the
maximumShiftCredibility function in BAMMtools.
To determine the effect of discrete trait evolution on diversifi-
cation within the Miconieae and to estimate the rates of transi-
tion between character states, we implemented the binary state
speciation and extinction (BiSSE) approach pioneered by Mad-
dison et al. (2007). BiSSE is a likelihood-based approach that
models trait evolution simultaneously with lineage diversifica-
tion within a phylogeny, and it is a useful tool for testing hypoth-
eses about the effects of biological characteristics of organisms
on speciation and extinction rates (Maddison et al. 2007;
O’Meara and Beaulieu 2016). We followed methods proposed
by Beaulieu and O’Meara (2016) to mitigate the high “false pos-
itive” rate of BiSSE methods by comparing trivial null BiSSE
models with the more complex null hidden state speciation
and extinction (HiSSE) models (R package HiSSE; Beaulieu
and O’Meara 2016) for each binary character in the matrix. If
the trivial null BiSSE model had a better fit on the basis of the
Akaike information criterion (AIC), we generated a suite of
eight BiSSE models of varying complexity (make.bisse in diver-
sitree; FitzJohn 2012), assuming that probability of sampling is
independent of character states, with a sampling fraction of 0.19
for each state to account for unsampled Miconieae species (sup-
plementary material VI). We then sampled the parameter space
of the best-performing model, chosen using AIC, with Markov
chain Monte Carlo for 100,000 generations with an exponen-
tial prior. We also performed the same series of analyses for
the categorical characters with more than two states: pore size
and anther symmetry. These characters were converted to binary
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according to their biological function (broad and rimose pores
were grouped together as opposed to minute pores; asymmetric
and monosymmetric anthers were grouped together as opposed
to polysymmetric anthers).
Results
The phylogenetic relationships of 1167 species were recon-
structed using maximum likelihood so that the evolution of floral
morphology could be studied (time-calibrated phylogeny avail-
able in supplementary material VIII). Sampling spanned 10 clades,
following the taxonomic assignments of Goldenberg et al. (2008).
The phylogenetic tree was used as input for all downstream anal-
yses, but it is not the emphasis of this study, and thus we will not be
discussing the taxonomic implications of these findings. Instead,
we focus on implications for the evolutionary history of morpho-
logical characters.
Examination of field photographs of more than 350 Mico-
nieae species has confirmed the striking variability in floral traits
for a tribe with conserved flower morphology (app. 2A, 2B; sup-
plementary material VII; fig. 1). Petal and anther colors varied
among white, yellow, and either pink or purple, which allowed
for many different color combinations across the tribe. Variabil-
ity of the shapes, positions, and sizes of reproductive organs
allowed for various phenotypes, with variation in forms of sym-
metry. While many species had herkogamous flowers, the same
effect (separation between stigma and anthers) was achieved
through various combinations of anther and stigma positions,
shapes, and lengths (compare fig. 1F, 1J, 1L).
Ancestral State Reconstructions and Variation
in Floral Morphology
Ancestral state reconstructions provided insight into the pos-
sible phenotype of the ancestral flower of Miconieae. The most
recent common ancestor of all Miconieae likely had flowers
with white petals and colorful (either yellow or pink, blue, or
purple) anthers on the basis of the probabilities of these states
at the node representing the most recent common ancestor of
Miconieae (93% probability of white petals; combined 59%
probability of colorful anthers; fig. 2). The probability of pink
or yellow being the ancestral state of petal color was much lower
(pink petals: 7%; yellow petals: 0%). The ancestral anther color
with the highest probability was yellow (47%), followed by
white (41%) and pink or purple (12%). The style of the ances-
tral flower was reconstructed as curved and slanted away from
the central axis (curved style: 63%; slanted away from flower
axis: 56%; fig. 3). Anthers of the ancestral flower probably
had minute pores and were monosymmetrically arranged (mi-
nute pores: 98%; monosymmetry: 77%; fig. 4).
Visual inspection of character states mapped on the phylogeny
along with ancestral state reconstructions indicates that white
petals were the most common, while pink petals evolved from
white ones several times throughout the evolutionary history of
Miconieae (fig. 2A). Although pink petals were distributed through-
out the phylogeny (clades Conostegia, Caribbean, Tococa, Cen-
trodesma, Secundiflorae, some Clidemia), they constituted only
a quarter of the species studied, and only two clades (Miconia
thomasiana clade within the Caribbean Miconieae and Tococa)
 GAVRUTENKO ET AL.—MORPHOLOGY AND SYMMETRY IN MICONIEAE FLOWERS 000

Fig. 1 Floral diversity within Miconieae, exemplified by species with variable floral traits. A, Clidemia capitellata. B, Clidemia ciliata.
C, Charianthus purpureus. D, Maieta poeppigii. E, Anaectocalyx bracteosa. F, Leandra australis. G, Miconia ampla. H, Miconia cernua.
I, Mecranium haemanthum. J, Conostegia macrantha. K, Miconia affinis. L, Pachyanthus pedicellatus. M, Miconia cerasiflora. N, Miconia
aureoides. O, Miconia brachycalyx. P, Miconia corymbiformis. Scale bars p 5 mm.

exhibited predominantly pink petals. White petals were present
in almost three-quarters of all species studied and were repre-
sented in nearly every clade within the phylogeny. Species within
two large clades (Miconia and Cremanium) had predominantly
white petals. Yellow petals were very rare and appeared in only
six species (two closely related species within Miconia, one in
Caribbean, and three in Cremanium; fig. 1H, 1P). Yellow petals
seem to have evolved from white ones.
Anther color was a more labile character than petal color.
Most of the clades contained all three phenotypes, although
pink, purple, or blue anthers were less frequent (fig. 2). White
anthers were more common in Miconia (fig. 1K) and Clidemia
(fig. 1A), while nearly all species within Caribbean Miconieae
and Conostegia had yellow anthers (fig. 1J). Ancestral state re-
constructions suggested that white anthers had evolved from
colorful anthers (either pink or yellow).
Deviations from polysymmetry in the gynoecium were fre-
quent within the tribe (fig. 3). A straight style evolved from a
curved style in several instances. Most species within Cremanium
and Clidemia had flowers with a straight style (fig. 3B), which

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000 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 2 Ancestral state reconstructions of petal color (A) and anther color (B) in Miconieae. Character states of the terminal branches are at the
tips. Node labels show ancestral state probabilities. Taxonomic assignment of species is indicated by arches with clade numbers: (1) Cremanium,
(2) Caribbean, (3) Conostegia, (4) Mecranium, (5) Miconia, (6) Clidemia (*Leandra), and (7) Tococa 1 Secundiflorae 1 Centrodesma.

could be either slanted away from the floral axis or directed up-
ward. However, an upward style was less frequent, and only the
Cremanium clade mainly contained species with an upward style
(fig. 3A). Most of the species in the phylogeny had styles that were
directed away from the floral axis at an angle starting at the ovary.
Anther symmetry varied between polysymmetric, monosym-
metric, and asymmetric; polysymmetric anthers (68% of all species)
were more common than anthers with deviations from poly-
symmetry (fig. 4B). Asymmetric anthers appeared in several spe-
cies and seem to have evolved from the monosymmetric character

Fig. 3 Ancestral state reconstructions of style direction (A) and style curvature (B) in Miconieae. Character states of the terminal branches are
at the tips. Node labels show ancestral state probabilities. Taxonomic assignment of species is indicated by arches with clade numbers: (1) Cre-
manium, (2) Caribbean, (3) Conostegia, (4) Mecranium, (5) Miconia, (6) Clidemia (*Leandra), and (7) Tococa 1 Secundiflorae 1 Centrodesma.

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 GAVRUTENKO ET AL.—MORPHOLOGY AND SYMMETRY IN MICONIEAE FLOWERS 000

Fig. 4 Ancestral state reconstructions of pore size (A) and anther symmetry (B) in Miconieae. Character states of the terminal branches are at
the tips. Node labels show ancestral state probabilities. Taxonomic assignment of species is indicated by arches with clade numbers: (1) Cremanium,
(2) Caribbean, (3) Conostegia, (4) Mecranium, (5) Miconia, (6) Clidemia (*Leandra), and (7) Tococa 1 Secundiflorae 1 Centrodesma.

state. Polysymmetric anthers evolved from monosymmetric ones in
Cremanium (fig. 1H, 1P), Conostegia, and Mecranium (fig. 1I) and
from asymmetric ones in Clidemia (fig. 1B, 1D, 1F) and Miconia
(fig. 1K).
Anther pore sizes varied between minute, broad, and rimose
(slits), with minute pores being the most common (74% of all
species; fig. 4A). Broad pores evolved within Miconia but ap-
peared in only a few species, while Cremanium mainly con-
tained species with broad anther pores. Ancestral state recon-
structions indicated that the rimose pore phenotype (slits) did
not evolve from minute pores but from broad pores.
Flower size varied greatly within the tribe, with an evolutionary
trend toward smaller flowers occurring in Cremanium, Miconia,
and Leandra and toward larger ones in Tococa (fig. 5). Petals of
some species were only 1 mm in length, while other species dis-
played large petals reaching a length of 20 mm (fig. 5A; app. 2A;
supplementary material VII). Anther sizes also fell within a wide
range, from 0.4 to 10 mm long (fig. 5B; app. 2A; supplementary
material VII).
and petals were white; fig. 1K, 1P). Such a phenotype was found
in 15% of all species and appeared in multiple instances across
three clades (Miconia, Cremanium, and Mecranium). These
findings were supported by Pagel’s correlation test, which
showed significant correlation between 10 out of 15 pairs of ex-
amined binary characters (table 1). On the other hand, Pagel’s
test indicated that herkogamy, though found in most species,
was correlated only with pore size and style direction.
Regression analysis of phylogenetically independent contrasts
showed a significant correlation between anther and petal sizes
(P  p 2:2 # 10–6 , r2 p 0:506), with a curious deviation in
some clades within Clidemia and Miconia. A map of continuous
traits plotted against each other shows that petal and anther sizes
covary, with integrated evolutionary trends in floral size (fig. 5).
In Clidemia and several subclades of Miconia, petal size de-
creased or remained the same with a simultaneous increase in an-
ther length, resulting in a peculiar floral phenotype with long,
often colorful anthers and small, pale, and often reflexed petals
(fig. 1B, 1F, 1G, 1K).

Trait Correlation Diversification Analyses

Correlations between discrete characters were assessed with a
Pagel’s test for correlated trait evolution and through visual in-
spection of character states mapped over the phylogeny. Pore
size was correlated with symmetry in both the androecium
and the gynoecium as well as with contrast between the petals
and anthers of the flower. This was particularly evident when
broad and rimose pores were grouped together so that pore size
could be converted into a binary character (two character states:
minute pores and large pores). Large pores were more common
in flowers with polysymmetric anthers, a straight upward style,
and a lack of anther/petal contrast (especially when both anthers
Diversification rates within the entire tribe (1083 species) with
one outgroup (four species of Eriocnemeae; sensu Penneys et al.
2020) were modeled with BAMM. There was a notable hetero-
geneity in diversification rates through time and among different
clades. The maximum shift credibility configuration with the max-
imum marginal probability of rate shifts along branches detected
nine diversification rate shifts (fig. 6). The oldest shift occurred
approximately 17 mya, near the most recent common ancestor
of the tribe Miconieae, and denotes the split between the clade of
Eriocnema and Physeterostemon (four samples in this phylogeny
out of seven known species) and the radiation of the Miconieae

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000 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 5 Map of trait evolution of maximum petal lengths (A) and anther lengths (B; log transformed). Trait values of terminal branches and
estimated trait values of internal nodes are shown with a color gradient. Terminal branches are aligned for a perfect match between species in
the phylogenies. Species names are omitted for clarity. Clade assignments: (1) Cremanium, (2) Tococa, (3) Clidemia, and (4) Miconia.

(fig. 6; shift 1). The second shift occurred 12 mya at the node
representing the most recent common ancestor of a clade that in-
cludes Conostegia, Cremanium, and the Caribbean clades (shift 2).
Diversification rates within this group increased and remained
higher than those of the rest of the tribe, except for clades with
more recent diversification shifts. The other seven diversification
shifts occurred later, between 5.3 and 1 mya. Two shifts in Cre-
manium (2.5 and 4 mya; shifts 4 and 5), one shift in Clidemia
(5.3 mya; shift 6), and a shift in Miconia (4 mya; shift 9) exhibit ini-
tial increases in diversification rates followed by slight decreases.
A shift in Mecranium (1 mya; shift 3) corresponds to a higher di-
versification rate without a recent rate decrease. Leandra experienced

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 GAVRUTENKO ET AL.—MORPHOLOGY AND SYMMETRY IN MICONIEAE FLOWERS
Table 1
Results of Pagel’s Test for Correlated Evolution of Discrete Characters
Contrast Anther symmetry
Pore size 7.8 # 10 *27
.0003*
Contrast .0008*
Anther symmetry
Style curvature
Style direction
Note. Numbers are P values. Significant results are indicated by asterisks.
two consecutive diversification shifts. The first one (4 mya; shift 7),
near the origin of the clade, resulted in a slight increase in di-
versification rate and was followed by a burst (1.5 mya; shift 8)
toward the highest diversification rates within the entire Mico-
nieae tribe.
A suite of models generated for every trait under BiSSE and
HiSSE scenarios provided evidence for state-dependent diversi-
fication in only one trait, herkogamy (table S2). The best model
for herkogamy had equal speciation rates for herkogamous and
nonherkogamous flowers (table S3). While the rates of transi-
tion toward herkogamy were significantly greater than the re-
verse (fig. 7A), plants with nonherkogamous flowers showed
lower extinction rates (fig. 7B).
Discussion
Examination of floral trait evolution using the largest species-
level phylogeny of Miconieae to date with morphological data
on a variety of floral characteristics for more than 350 species
revealed rampant convergent and correlated evolution in most
floral characters.
We detected correlated evolution of multiple characters indic-
ative of pollinator-mediated selective pressures and confirmed
parallel evolutionary trends toward phenotypic generalization
previously suggested for this phylogenetically complex tribe (Gol-
denberg et al. 2008; Varassin et al. 2008; Brito et al. 2016). In
our analyses, we did not set out to infer the pollinator from mor-
phology but rather investigated which character states tend to co-
occur when the floral phenotype deviates from what we know to
be a specialized phenotype in this tribe, such as the proposed an-
cestral phenotype.
Ancestral Flower
We conclude that the ancestral flower likely had white petals
and colorful anthers and displayed monosymmetry in both the
androecium and the gynoecium. It was likely herkogamous,
had a curved style directed away from the center of the flower,
and had monosymmetric stamens with minute anther pores
(see fig. 1G, 1L for such color and shape combinations). Such
floral features are commonly observed in Melastomataceae, al-
though yellow anther color is more widespread than pink or
purple (Renner 1989; Larson and Barrett 1999). This phenotype
is consistent with morphological specialization toward buzz
pollination by bees, which is the predominant mode of pollina-
tion in the family and is ancestral in Miconieae (Buchmann
1983; Renner 1989). Across angiosperm clades, minute anther
pores in buzz-pollinated flowers are commonly interpreted to
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000
Style curvature Style direction Herkogamy
1 2.3 # 217
10 * 1.1 # 10205*
.0104 1.4 # 10206* .937
1.8 # 10218* 2.2 # 1027* .560
1.7 # 1025* .449
9.2 # 1026*
be an adaptation to prevent the release of concealed pollen by
illegitimate flower visitors (Buchmann 1983; Marazzi et al.
2007; Arceo-Gómez et al. 2011; Mesquita-Neto et al. 2018).
Similarly, the evolution of monosymmetry in the androecium
and gynoecium is often associated with shifts toward specialized
pollination, as these functional modules are responsible for pol-
linator orientation and precise pollen placement (Neal et al.
1998; Pérez et al. 2006; Kay and Sargent 2009; O’Meara et al.
2016).
Morphological Diversity
Miconieae is a species-rich tribe that exhibits great floral diver-
sity, with a great amount of variation in individual floral traits,
which jointly produce a wide range of floral morphologies. Ex-
istence of such variability in floral traits despite constraints im-
posed by the conserved floral plan supports the idea that, rather
than being balanced on an “adaptive peak” toward highly suc-
cessful bee buzz pollination (Renner 1989), Melastomataceae
are indeed wandering an adaptive plateau of very different mor-
photypes exploiting the same syndrome (Reginato and Michel-
angeli 2016; Dellinger et al. 2019a). Such phenotypic diversity
also demonstrates a rich canvas for various pollinator interac-
tions and thus multiple opportunities for departures from the
syndrome. We discuss individual trait variability and evolution-
ary trends inferred from ancestral trait reconstructions to gauge
whether they can be viewed through the prism of adaptation to-
ward pollinator morphology and behavior on the specialization-
generalization spectrum.
Petal Color
Petal color is an important visual cue for both insect and bird
pollinators (Renner 1989; Lunau 2006) but is informative for
inferences about pollination mode only when viewed in combi-
nation with other characters. For instance, there is plenty of ev-
idence for the role of petal color in bee signaling to interpret
emergence of colorful petals in some Miconieae as adherence
to the specialized buzz-pollination mode. Colorful petals attract
bees from a distance and are important in providing a con-
trasting background for anthers as a short-distance cue (Larson
and Barrett 1999; Heuschen et al. 2005; Lunau 2006). Alterna-
tively, pink petals could indicate shifts to a different specialized
pollination mode by hummingbirds, especially when brightly
colored pink or deep-red petals form a tubular shape in a mature
flower (Varassin et al. 2008). Such pollination has been ob-
served in several nectariferous species of Miconieae (Penneys
and Judd 2003, 2005; Goldenberg et al. 2008; e.g., see Cha-
rianthus purpureus in fig. 1C). Yellow petals were observed in
000 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 6 Diversification rates of Miconieae and Eriocnemeae estimated with Bayesian analysis of macroevolutionary mixtures. Diversification rates
were estimated for a phylogeny of 1083 species of Miconieae, three species of Physeterostemon, and one species of Eriocnema (all other outgroups
removed) and are shown with a color gradient (number of lineages per million years). Clade assignment is indicated by bars on the right. Circles denote
the diversification rate shifts with the maximum marginal probability. The bar on the bottom shows a geological timescale in millions of years.

only a few species, and they often either were short and incon-
spicuous or lacked anther/petal contrast, suggesting that yellow
pigment in these species would not be an effective attractive cue
for bee pollinators. However, as a contrasting backdrop to
brightly colored anthers (Lunau 2006) or in shaded flowers of
understory plants (Renner 1989), white petals could also pro-
vide a strong visual signal to bees. We also observed some strik-
ing floral displays created by white petals of small flowers ar-
ranged in dense inflorescences, although inflorescence size or
structure was not formally included in our analyses. Conversely,

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 GAVRUTENKO ET AL.—MORPHOLOGY AND SYMMETRY IN MICONIEAE FLOWERS
Fig. 7 Trait-dependent diversification in Miconieae. A, Transition
rates between character states under the best binary state speciation
and extinction (BiSSE) model of herkogamy evolution (speciation
rates are constant). B, Extinction rates of character states under the
best BiSSE model of herkogamy evolution (speciation rates are con-
stant). Marginal distributions for transition and extinction rates were
sampled with Markov chain Monte Carlo. Horizontal bars show 95%
confidence intervals.
retention of white petals could be evolutionarily favored by gen-
eralist pollination modes where bright petal pigment no longer
functions in pollinator attraction and could be selected against
as an unnecessary expense—especially if an alternative reward
is offered. In multiple documented cases of generalized pollina-
tion systems in Miconieae, when a nectar reward supplements
or substitutes a pollen reward, petal color is white (Goldenberg
and Shepherd 1998; Varassin et al. 2008; Kriebel and Zumbado
2014; Brito et al. 2017). Moreover, while only a few studies that
looked at Melastomataceae flowers have expressly addressed
the visual color ability of bees (Buchmann and Buchmann
1981; Gross 1993; Velloso et al. 2018), some weak UV patterns
were found in a species of Memecyleae. This is an important vi-
sual guide for bee pollinators (Lunau 2000), and corolla and an-
droecium colors on the UV spectrum should be thoroughly in-
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000
vestigated. Additionally, there is a possibility of nonadaptive
evolution of this trait due to genetic correlation with other floral
or vegetative traits (Armbruster 2002), and thus caution is nec-
essary when petal color is considered through the prism of pol-
linator interactions. We conclude that, although petal color
provides important context for the inference of shifts in pollina-
tion modes, such interpretation is strongly dependent on the
states of other character traits and should be analyzed jointly
with corolla shape and androecium morphology as well as pol-
linator reward and inflorescence architecture (two traits not in-
cluded in this study).
Anther Color
We attribute the observed anther color variability and multi-
ple historic changes in this trait to the role of anthers in floral sig-
naling. In buzz pollination, the pollen is not visible to the polli-
nator, and the anther itself assumes the signaling function to
attract pollinators seeking a pollen reward (Lunau 2006). Yel-
low anthers in particular mimic the color of pollen and trigger
innate responses in pollen-collecting bees and flies (Lunau and
Wester 2017; Lunau et al. 2017), although bees have been
reported to instinctively respond to colors other than yellow
and to demonstrate learning behavior (Lunau 2006). In addi-
tion, removal of yellow anthers, stamens, or stamen appendages
in species of Melastomataceae resulted in reduction of visitation
by bees (Larson and Barrett 1999; Luo et al. 2008). Multiple
studies of Melastomataceae and other angiosperm families asso-
ciated evolution of elaborate anthers, including evolution of
heteranthery, where anthers are organized in separate “pollinat-
ing” and “feeding” functional whorls of different size, shape,
and color (Luo et al. 2008; Vallejo-Marín et al. 2009, 2014),
with adaptation to buzz pollination (Marazzi et al. 2007; Luo
et al. 2008; Vallejo-Marín et al. 2010). Interestingly, such divi-
sion of labor was not observed in Miconieae, although it is fre-
quent in sister tribes within Melastomataceae (Vallejo-Marín et al.
2010). We consequently interpret the retention of yellow anthers
in every major clade of Miconieae as a consequence of ancestral
specialized pollinator modes, whereas the repeated evolution of
white anthers might be indicative of generalized pollination. We
conclude that, as a short-distance signaling cue for pollinators,
anther color is an important and more reliable predictor of pol-
lination mode than petal color.
Anther Pore Size
Consistent with previous findings by Brito et al. (2016), we de-
tected convergent evolution of broad and slit-like pores within
Miconieae, and we interpret such pore broadening as an evolu-
tionary trend toward generalization on the basis of the functional
differences between pollen extraction from anthers with large
pores and from anthers with minute pores. Departures from buzz
pollination required the evolution of alternative mechanisms of
pollen expulsion (“bellows” and “saltshaker” modes) and pollina-
tor reward in Merianieae (Dellinger et al. 2019a, 2019b). In
Miconieae, the evolution of broad pores allows pollen expulsion
by nonvibratory animals and is in some cases accompanied by nec-
tar production (Goldenberg and Shepherd 1998; Varassin et al.
2008; Kriebel and Zumbado 2014). Such increase in the pore size
is thought to have adaptive value because, with a supplementary
000 INTERNATIONAL JOURNAL OF PLANT SCIENCES
nectar reward, it seems to expand the number of pollinator spe-
cies without excluding buzzing bees (Goldenberg et al. 2008;
Varassin et al. 2008; Kriebel and Zumbado 2014). This is con-
sistent with the retention of ancestral pollinators as secondary
pollinators and the existence of intermediate phenotypes adapted
to both ancestral and derived pollinators in floral syndrome
transitions in angiosperms (Pérez et al. 2006; Wilson et al.
2006; Rosas-Guerrero et al. 2014). Further investigation is nec-
essary to determine how widespread nectar rewards in broad-
pored Miconieae species are.
Symmetry
Nearly identical patterns of historic diversification in androe-
cium and gynoecium symmetry demonstrate the integrated nature
of floral function. Changes in floral symmetry are often observed
in pollinator shifts, although the evolution of monosymmetry as
an adaptation to specialized pollinators, including adaptations
toward buzz pollination, is more commonly reported (Marazzi
et al. 2007; Jabbour et al. 2008; Arceo-Gómez et al. 2011;
O’Meara et al. 2016; Vamosi et al. 2018). Such linked patterns
of symmetry in reproductive organs are necessary for precise
pollinator orientation and pollen extraction, successful pollen
transfer, and preventing unmatched visitors from pollinating
the flower (Larson and Barrett 1999; Luo et al. 2008; Vallejo-
Marín 2019). In such specialized flowers, monosymmetric posi-
tions and the shapes of the style and anthers would be the evo-
lutionary results of adaptation to the most effective pollinator
species and could thus be interpreted as indicative of a special-
ized pollination mode (De Luca and Vallejo-Marín 2013). How-
ever, polysymmetric buzz-pollinated morphologies, including
the classic Solanum flower phenotype, are also common (De
Luca and Vallejo-Marín 2013).
Besides pollinator fit, variations in stamen symmetry, com-
bined with the shape and direction of the style, can also be
interpreted from the point of view of herkogamy, which is the
main mode of outcrossing promotion in the Melastomataceae
(Renner 1989). Herkogamy seems to be evolutionarily favored
within the Miconieae, which would explain the multitude of di-
verse herkogamy types achieved in combinations of morpho-
logical features of several floral units. Although reduction of
herkogamy has been reported in species of Miconieae with gen-
eralized pollination syndromes (Kriebel and Zumbado 2014),
herkogamy could be conserved within the tribe because of the
reproductive advantages of outcrossing rather than as a result
of pollinator-mediated selection. Further investigation that ac-
counts for self-incompatibility and apomixis (reported but not
thoroughly studied in the tribe) is necessary to tease apart the
agents of adaptation in this trait (Goldenberg and Shepherd
1998; Maia et al. 2016).
Flower Size
We found some support for the previously reported conver-
gence toward floral size reduction in some Miconieae clades
(Goldenberg et al. 2008; Brito et al. 2016), although the results
could be an artifact of the ancestral reconstruction method for
continuous traits and should be interpreted with caution (Web-
ster and Purvis 2002). Adaptations toward bees of a certain size
were shown to preclude visitation by mismatched bee species in
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several study systems (Dalechampia [Euphorbiaceae]: Arm-
bruster 1988, 1990; Collinsia [Plantaginaceae]: Heinrich and Ra-
ven 1972; Armbruster et al. 2002). In Miconieae, decrease in size
expands the pollinator assemblage when flowers are grouped
into inflorescences, and therefore it can be seen as a generaliza-
tion strategy. In addition to large buzzing bees that can vibrate
and pollinate the entire inflorescences of small flowers, buzzing
bees that are too small to be effective pollinators of larger flowers
can also pollinate such flowers (Goldenberg et al. 2008). Broad-
pored flowers in dense inflorescences can further exploit pollina-
tion by a variety of nonbuzzing insects, and they warrant further
inquiry to determine whether the evolution of nectar reward
parallels the reduction in size. An additional advantage of size re-
duction in species that present pollinators with a “big bang” floral
display (flowering strategy with many flowers open at once) is
eliminating the need for the production of petal pigment for polli-
nator attraction (Kriebel and Zumbado 2014).
Trait Correlation and Character State Co-occurrence
Correlation analyses and examination of character state co-
occurrence exemplify that several traits should be considered
in unison to inform hypotheses about shifts in pollinator modes,
as inferring the taxon or even the functional guild of a flower’s
pollinator is quite difficult using any single character within our
morphological framework. Highly significant correlation among
floral traits demonstrates the integrated nature of floral function
and is well documented in animal-pollinated flowers (Endress
2006, 2016; Diggle 2014). As coordination of floral traits is re-
quired for successful pollination, their correlated evolution is
not surprising, especially in functional modules of traits respon-
sible for pollinator orientation and pollen deposition on the pol-
linator body and stigma (Ordano et al. 2008; Rosas-Guerrero
et al. 2010). Demonstrated in our study by the nearly identical evo-
lutionary patterns of symmetry changes in the androecium and
the gynoecium, interaction of such functional traits was found
to be more important for differentiating pollination modes than
that of floral traits involved in attraction in several recent studies
(Rosas-Guerrero et al. 2010; O’Meara et al. 2016; Dellinger et al.
2019a). In addition to great disparity in floral morphologies due
to different combinations of constituent traits, the modularity of
floral traits is further illustrated by the divergence of allometric
trajectories in the petal and anther size of some Miconia and
Leandra species. Such change in the proportion of floral parts,
specifically those involved in pollination, has been strongly asso-
ciated with pollinator-mediated selection (Kirchoff 1983; Ushi-
maru and Nakata 2001; Summers et al. 2015) and may be attrib-
uted to anthers assuming the function of pollinator attraction and
guidance in these clades.
The repeated co-occurrence of certain character states and the
convergent evolution of a (suspected) generalist flower pheno-
type demonstrate the need for synergistic approaches to studies
of floral evolution (Donoghue and Sanderson 2015; O’Meara
et al. 2016). Such a comprehensive view of diversification con-
siders the interplay of trait combinations that sequentially come
together, providing morphological context for trait transitions.
We found that a phenotype with broad anther pores, a straight
style, and lack of anther/petal contrast emerged multiple times
in the phylogeny, but it is never found in flowers with a mono-
symmetric androecium. This seems to suggest that the evolution
 GAVRUTENKO ET AL.—MORPHOLOGY AND SYMMETRY IN MICONIEAE FLOWERS
of a polysymmetric androecium was a prerequisite for subse-
quent transitions toward derived character states in the other
traits, in effect providing a path toward generalist pollination
and away from the adaptive plateau of buzz pollination. A sim-
ilar example of one character state hinging on the state of the
other is the evolution of nectar spurs conditional to the emer-
gence of monosymmetry in the Asteridae (Jabbour et al. 2008).
Diversification Analyses
The oldest shift in diversification dynamics detected with
BAMM corresponds to the diversification burst at the base of
the Miconieae clade first reported by Judd and Skean (1991)
and may be the reason for difficulties in morphological clade de-
limitation (Goldenberg et al. 2008; but see Berger et al. [2016]
for an unexpected alternative shift configuration). Similarly,
the second burst in diversification rates 12 mya may reflect the
early radiation events in the tribe with complex biogeographical
history (described in Michelangeli et al. 2008). Of the seven
more recent shifts in the tribe, five occur within Clidemia and
Cremanium, two clades that share some floral characteristics
but that likely diverge in their pollination modes. Species in both
clades exhibit predominantly polysymmetric anthers, white petals,
and straight styles (figs. 2B, 3B, 4B), but access to nonvibratory
visitors is restricted by minute pores in Clidemia and is enabled
by broad/rimose pores in Cremanium. Interestingly, the more re-
cent second diversification rate shift in Leandra occurred within
the clade Carassanae, which has greater floral variability and
more shifts between different morphological regimes in flowers
than the rest of Leandra (Reginato and Michelangeli 2016).
Such variability of floral phenotypes would broaden the assem-
blage of buzzing bee species able to pollinate the flowers in this
clade and may be indicative of adaptive evolution.
In addition to their morphology, Leandra and Cremanium
are distinct from the rest of the tribe because the representatives
of both clades are commonly found at higher elevations than
other Miconieae species (Goldenberg et al. 2008; Michelangeli
et al. 2008; Reginato 2016; Reginato and Michelangeli 2016).
This suggests that biogeography plays an important role in
shaping species diversification and pollinator interactions in
the tribe. Rapid diversification concurrent with parallel patterns
of floral evolution is likely a product of ecological opportunities
provided by the environmental heterogeneity observed in ele-
vated habitats (Hughes and Atchison 2015). Montane condi-
tions are also associated with shifts in pollinator mode due to
the unpredictability of pollinators at higher elevations (Cruden
1972; Varassin et al. 2008; Dellinger et al. 2014), although such
transitions usually involve specialized vertebrate pollination
(Pérez et al. 2006; Rosas-Guerrero et al. 2014). It appears that
shifts in Miconieae pollination strategies may also involve a
transition from specialized bee pollination to generalist pollina-
tion in elevated environments where buzz pollination is not re-
liable (Varassin et al. 2008; Brito et al. 2016). Further investiga-
tion into drivers of diversification in Miconieae should include
combinations of floral traits (O’Meara et al. 2016) viewed in
biogeographical context, as our analyses of individual traits
failed to detect state-dependent diversification that could fully
explain these shifts.
BiSSE analyses helped detect the role of herkogamy in Mico-
nieae diversification, leading to further questions about the dy-
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000
namics of floral morphology in the context of herkogamy and
pollination mode generalization. Physical separation between
the stigma and the anther pores is achieved through spatial ar-
rangement and the shapes of reproductive organs within the
flower, with nonherkogamous Miconieae flowers presenting
some of the same structural features found in flowers with a gen-
eralized pollination syndrome within the tribe: a polysymmetric
androecium with a straight, upright style. While the faster tran-
sition toward herkogamy is an expected consequence of the evo-
lutionary advantages of outcrossing, the lower extinction rate in
nonherkogamous species warrants further investigation. Loss of
herkogamy could lead to decreased extinction during coloniza-
tion of novel environments, where plant species capable of self-
fertilization have adaptive advantages over species with obligate
specialized pollinators that may be unavailable during range ex-
pansion (Toräng et al. 2017). Species colonizing species-poor
habitats devoid of specialized pollinators, such as newly emerged
mountain ranges, islands, or disturbed habitats, are more suc-
cessful if they are able to reproduce asexually or through self-
pollination (Sandvik et al. 1999; Kriebel et al. 2015), providing
one potential explanation for the greater success of nonherko-
gamous species. Alternatively, emergence of physiological self-
incompatibility could eliminate the need for herkogamy, while
such generalized nonherkogamous phenotypes would increase
the diversity of pollinators, resulting in greater reproductive suc-
cess. Self-incompatibility has been documented in Miconieae,
and Goldenberg and Shepherd (1998) suggest that nearly a quarter
of all Miconieae could be self-incompatible, but the current knowl-
edge of the issue in such a species-rich tribe is very limited. More-
over, further investigation of herkogamy in Miconieae should
include floral grouping, as potential decreases in intrafloral herko-
gamy in dense inflorescences were not accounted for in our study.
Conclusions
Our examination of the floral morphology of the Miconieae
confirms that multiple traits within the flowers evolve in a uni-
fied fashion because of the integrated functioning of these angio-
sperm structures. Correlated evolution of multiple floral traits
emphasizes the need for consideration of trait combinations in
studies that focus on the processes that affect reproductive trait
morphology. We detect analogous trends toward generalized
pollination in a range of characters related to the size, color,
shape, and disposition of reproductive structures within the
Miconieae flowers. We find an association between generaliza-
tion trends and increased diversification rates that may be re-
lated to adaptations to highland environments within the tribe.
Our analysis of the general evolutionary trends within the tribe
provides a guide for future targeted investigations of various
aspects of floral morphology within this study system. More
field studies are necessary to determine pollinator assemblages
of species with various morphotypes and the incidence of alter-
native rewards and to expand our knowledge of self-compatibility
within the tribe. Additionally, future analyses would need to
consider other aspects of floral displays, for instance, inflores-
cence size and architecture as well as UV patterns in flowers. Fi-
nally, exploration of herkogamy and the morphological dispar-
ity of individual traits (especially anther shape and pore size)
and morphotypes could benefit from thorough quantification
using 3D morphometrics (van der Niet et al. 2010).
000 INTERNATIONAL JOURNAL OF PLANT SCIENCES
Acknowledgments
This work was possible thanks to the generosity of many
colleagues who have shared photographs of plants in the field
and/or plant tissue, most notably, Renato Goldenberg, Walter S.
Judd, Gretchen Ionta, Lucas Majure, Darin S. Penneys, Frank
Almeda, Marcela Alvear, Carmen Ulloa, Eldis R. Bécquer,
Mayara K. Caddah, John L. Clark, Ken Wurdack, Karen Red-
den, and Mac Alford. We thank Mathew Sewell and the staff
at Pfizer Plant Research Laboratory of the New York Botanical
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