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American Journal of Botany 106(7): 1021–1031, 2019; http://www.wileyonlinelibrary.com/journal/AJB © 2019 Botanical Society of America • 1021
e co‐occurrence of closely related plant species in the same com-
munity poses a challenge for species that share similar habitats or
resources. At sympatric sites, congeneric species may not only com-
pete for abiotic resources and pollinators, but they are also vulnera-
ble to reproductive interference (i.e., interspecic reproduction that
reduces the tness of at least one of the coexisting species; Kyogoku,
2015). e phenotypic traits that prevent reproductive interference
may evolve in allopatry or sympatry, but in coexisting popula-
tions of congeneric species, they may act to prevent interspecic
gene ow and gamete wastage and the concomitant costs of hybrid
progeny production (Hopkins and Rausher, 2012). When interspe-
cic pollination represents a cost to reproduction, mechanisms of
reproductive isolation (RI) allow congeneric species to coexist with
reduced reproductive interference (Coyne and Orr, 2004).
Reproductive isolation barriers generally evolve during specia-
tion or upon secondary contact between recently diverged species. In
plants, these are divided into pre‐ and post‐pollination mechanisms
according to their timing of action within the reproductive cycle of
the plant (Baack etal., 2015). Pre‐pollination mechanisms preclude
interspecic pollen ow through changes in habitat use, phenology,
oral traits, and breeding systems, and they may evolve in allopatry
or under selection against unviable or unt hybrid progeny (Levin,
2006; Sobel et al., 2010). Post‐pollination mechanisms of repro-
ductive isolation generally result from the accumulation of genetic
Reproductive isolation among three sympatric Achimenes
species: pre‐ and post‐pollination components
Erandi Ramírez‐Aguirre1,2, Silvana Martén‐Rodríguez1,5 , Gabriela Quesada‐Avila3, Mauricio Quesada1,4, Yesenia Martínez‐Díaz1, Ken Oyama1,
and Francisco J. Espinosa‐García4
RESEARCH ARTICLE
Manuscript received 4 December 2018; revision accepted 6 May 2019.
1 Laboratorio Nacional de Análisis y Síntesis Ecológica
(LANASE),Escuela Nacional de Estudios Superiores (ENES),Unidad
Morelia,Universidad Nacional Autónoma de México, Morelia,
Michoacán C.P. 58190, México
2 Posgrado en Ciencias Biológicas,Universidad Nacional
Autónoma de México. Unidad de Posgrado,Coordinación del
Posgrado en Ciencias Biológicas. Edicio D,1º Piso. Circuito
de Posgrados,Ciudad Universitaria Del., Coyoacán C. P. 04510,
MéxicoD.F
3 Universidad Nacional de Costa Rica, Heredia, Costa Rica. Avenida1,
Calle 9. Apartado Postal, 86‐3000
4 Instituto de Investigaciones en Ecosistemas,Universidad Nacional
Autónoma de México, Antigua Carretera a Pátzcuaro 8701, Morelia,
Michoacán C.P. 58190, México
5 Author for correspondence (e‐mail: smartenr@gmail.com)
Citation: Ramírez‐Aguirre, E., S. Martén‐Rodríguez,
G. Quesada‐Avila, M. Quesada, Y. Martínez‐Díaz, K. Oyama, and
F. J. Espinosa‐García. 2019. Reproductive isolation among three
sympatric Achimenes species: pre‐ and post‐pollination components.
American Journal of Botany 106(7): 1021–1031.
doi:10.1002/ajb2.1324
PREMISE: Closely related species occurring in sympatry may experience the negative
consequences of interspecic pollen transfer if reproductive isolation (RI) barriers are not
in place. We evaluated the importance of pre‐ and post‐pollination RI barriers in three
sympatric species of Achimenes (Gesneriaceae), including ecogeographic, phenological,
oral isolation, self‐pollination, and hybrid viability (fruit and seed set).
METHODS: We recorded geographic distribution throughout species ranges and assessed
owering phenology and pollinator visitation at one site in central Mexico. In the
greenhouse, we measured oral traits involved in RI and quantied fruit and seed set for
from self, intraspecic, and interspecic crosses.
RESULTS: Ecogeographic barriers were important in RI, but under sympatry, phenological
and oral barriers contributed more to total RI. Phenological RI varied between species
and years, while oral RI was 100% eective at preventing interspecic visitation. Species
showed dierences in oral morphology, color, and scents associated with specialized
pollination systems (A. antirrhina–hummingbirds, A. ava–bees, A. patens–butteries);
heterospecic visitation events were restricted to rare secondary pollinators. Hybrid
crosses consistently yielded progeny in lower numbers than intraspecic crosses.
CONCLUSIONS: This study indicated that neither autogamy nor early post‐pollination
barriers prevent interspecic pollen ow between Achimenes species. However, oral
isolation, acting through a combination of attraction and reward traits, consistently
ensures specicity of the pollination system. These results suggest that selection on oral
traits to reduce the costs of hybrid progeny production may have played a role in evolution
or maintenance of specialized pollination systems in Achimenes.
KEY WORDS oral isolation; Gesneriaceae; hybridization; pollination; reproductive
interference; sympatry; temporal isolation.
1022 • American Journal of Botany
dierences during the process of speciation, and they act upon hy-
brid progeny production, viability, or performance (Orr and Turelli,
2001; Coyne and Orr, 2004). ese later barriers have reproductive
costs because gametes are wasted and energy is invested in unt
hybrid progeny; thus, pre‐pollination RI mechanisms are thought
to evolve rst and be stronger than post‐pollination barriers (Lowry
etal., 2008; Baack etal., 2015). However, more studies that jointly
evaluate both types of barriers are necessary to determine the prev-
alence and strength of the dierent forms of reproductive isolation
in lineages of closely related species (Baack etal., 2015).
Habitat isolation is a reproductive barrier caused by local adap-
tation and limited gene ow among allopatric populations, and it
may be one of the rst reproductive barriers to evolve inthe pro-
cesses of speciation (Sobel etal., 2010). In the last decade, ecogeo-
graphic isolation has been tested using dierent approaches (e.g.,
Ramsey et al., 2003; Kay, 2006), but only recently, new methods
based on ecological niche modeling have tested whether dierences
in geographic distributions might also reect changes in adaptation
to particular abiotic environments (Sobel, 2014). When species are
not geographically isolated, natural selection should favor the evo-
lution of alternative RI mechanisms.
In some plant groups, owering phenology plays an important
role at preventing interspecic pollen transfer because divergent or
sequential owering times among sympatric congeners allow the
temporal separation of pollinator use (e.g., Levin and Anderson,
1970). However, owering phenology oen varies between individ-
uals, populations, and years (Kudo, 2006). Furthermore, within a
plant lineage, phenology could be under ecological, developmental,
or historical constraints that might restrict variation in the timing
of reproductive events (Kochmer and Handel, 1986). In these cases,
oral traits related to pollinator attraction may contribute more to
reproductive isolation.
Floral isolation may occur through the mechanical coupling
between owers and pollinators or through oral traits that inu-
ence pollinator attraction and behavior (Grant, 1994). Specically,
the morphological t between owers and pollinators should max-
imize the precision of contact between oral sexual organs and an-
imal body parts, reducing interspecic pollen transfer (e.g., Pauw,
2006). Floral attractants and rewards also inuence pollinator pref-
erences and oral visitation, driving ethological isolation through
specic mechanisms of pollen transfer or pollinator constancy (e.g.,
Schemske and Bradshaw, 1999). e individual contributions of
traits involved in oral isolation vary among plant species (Lowry
etal., 2008); therefore, it is important to dissect the contribution of
dierent traits to RI.
Changes in plant breeding system have also been associated with
the prevention of interspecic pollen transfer in a number of plant
groups (e.g., Bromeliaceae, Matallana etal., 2010; Centaurium, Brys
etal., 2016). Early deposition of self‐pollen on stigmas may interfere
with deposition of outcross and heterospecic pollen (Brys etal.,
2016). A high potential for self‐pollination in addition to high levels
of natural seed production would indicate that autogamy might also
act as a barrier to interspecic pollen transfer in coexisting assem-
blages of closely related species. Although this topic has received re-
cent attention (Goodwillie and Ness, 2013; Briscoe‐Runquist etal.,
2014), information on autonomous self‐pollination as a mechanism
of RI is still sparse relative to our knowledgeof other forms of RI.
e family Gesneriaceae provides an interesting system to study
the dierent traits that contribute to reproductive isolation because
oral traits vary greatly and many species are habitat specialists
that coexist in sympatry (e.g., epiphytic and rupicolous species).
Furthermore, many Gesneriaceae species can produce hybrid prog-
eny through horticultural practices and in natural conditions (e.g.,
Qiu etal., 2011; de Villiers etal., 2013; Smith etal., 2017). Wiehler
(1983) proposed that pollinator specialization was one of the main
reproductive barriers responsible for maintaining the identity of
sympatric congeneric species of Gesneriaceae; however, this idea
has not been tested yet.
e aim of this study was to evaluate the importance of dier-
ent traits involved in the prevention of reproductive interference
among three sympatric Achimenes species from Mexico. Most
members of the genus Achimenes occur on wet riparian clis in
seasonal environments, creating a patchy mosaic of co‐existing
congeners. While hybrids are commonly generated for the orna-
mental plant market, hybrids in nature are rare (Ramírez‐Roa,
1987; E. Ramírez‐Aguirre etal., unpublished data). Achimenes
is a genus of recent evolutionary origin (Roalson and Roberts,
2016); therefore, it is possible that genetic barriers between spe-
cies are not fully developed. We evaluated the following repro-
ductive barriers: (1) ecogeographic isolation, (2) phenological
isolation, (3) oral isolation and its components (morphology,
nectar production and chemical composition, scent production,
and pollinator visitation), (4) isolation by self‐pollination, and
(5) fruit set and seed production as measures of hybrid viabil-
ity. Barriers 1–5 are considered pre‐mating, whereas barrier 5 is
post‐mating and represents the cost of hybridization at the level
of progeny production.
MATERIALS AND METHODS
Study site
e study was conducted in 2013–2016 during the months of July‐
November at La Tzarazacua Community Park, Parque Nacional
Barranca del Cupatitzio, Uruapan, Michoacan, Mexico (19°25′11″–
19°26′24″N, 102°07′40″–102°04′20 ″W, 1400–1500 m a.s.l.). Total
annual rainfall ranges between 1500–2000 mm, and mean annual
temperature is 18–20°C (CONANP, 2006). At the study site, the
vegetation includes a combination of pine–oak and cloud forest
patches that go from early to mid‐late successional stages, inter-
mixed with patches of cattle pasture. Achimenes species grow on the
outcrops of basaltic rock that characterize the basin and canyons of
the Cupatitzio River (CONANP, 2006).
Study species and greenhouse collections
According to the phylogenetic analysis by Roberts and Roalson
(2018), Achimenes is a genus of Mesoamerican origin that com-
prises approximately 26 species. Achimenes antirrhina, A. flava,
and A. patens belong to a larger clade comprising 10 species, and
although these species are not supported as sister to one another,
they last shared a common ancestor approximately 4 million years
ago (Ma) (Roalson and Roberts, 2016); therefore, they are species of
recent divergence. All species are geophytes that inhabit rocky habi-
tats on wet riparian slopes. Geophytes are perennial plants that pro-
duce new shoots every spring from underground organs. In the case
of Achimenes, these shoots reproduce during the growing season
and then die, leaving underground rhizomes dormant through the
winter. Only two individuals of putative hybrid origin (intermediate
July 2019, Volume 106 • Ramírez‐Aguirre etal.—Avoidance of interspecific reproduction in Achimenes species • 1023
corolla shape and color) were found at the study site between A.
flava and A. patens.
Achimenes antirrhina is a short herb, 30–50 cm long with red‐
orange, tubular owers that have an inner yellow throat (Fig.1A).
Achimenes flava is an herb 20–40 cm long with narrow, bell‐shaped,
yellow corollas (Fig.1B). Achimenes patens is a small herb 10–30cm
long that has violet, narrow, tubular owers with an elongated
spur (Fig.1C). All three species are protandrous; anthers dehisce
on the rst day of anthesis, and stigmas are fully receptive on the
fourth day. ese species have bilobed or stomatomorphic stigmas
(Ramírez‐Roa, 1987).
Living specimens of the three study species were grown ingreen-
houses located at Escuela Nacional de Estudios Superiores, UNAM,
Morelia. All specimens were collected as rhizomes and grown in
pots with a 1:1:1 mix of sphagnum, rock, and organic matter. To
follow natural cycles, watering was started in April and ended in
November, when aerial shoots dry out. Rhizomes were kept dry in
their pots until the following spring.
Pre‐pollination RI: ecogeographic barriers
To assess the role of geographic and habitat isolation in overall RI
between the three Achimenes species, we analyzed ecogeographic
isolation as proposed by Sobel (2014). Using seven bioclimatic lay-
ers (four layers that described temperature, and three layers that
described precipitation) and a topographic wetness layer with a
resolution of 1 km2, we modeled species distributions in MaxEnt
(Phillips and Dúdik, 2008). Using the resulting model layers, we
calculated ecogeographic isolation (Eco i) (RI2 equation of Sobel,
2014; see Appendix S1 for full equations and Appendix S2 for full
description of species distribution models). In summary, the equa-
tion for each species pair was: Eco i = 1 – [shared areas / (shared
areas + unshared areas for focal species)].
Pre‐pollination RI: owering phenology
e owering phenology of A. antirrhina, A. flava, and A. patens
was studied during 2014 and 2015 at La Tzararacua Community
Park. In 2014, we marked 60 plants of A. antirrhina, 51 of A. flava,
and 48 of A. patens. In 2015, we marked 50 plants of each species.
We counted the number of ower buds, open owers, and mature
fruits on each plant every 2 weeks from July to November each year.
Since not all plants survived, nal sample sizes might dier from
initial ones. For this reason, the number of owering individuals
was divided by the number of live individuals on each sampling
date.
Dierences in owering phenology between species were tested
using circular statistics and the circular package (watson.williams.
test function; Agostinelli and Lund, 2017) in R version 3.3.1 (R Core
Team, 2018). Circular statistics are appropriate for time data that
have an underlying cyclical distribution (Zar, 2014). e Watson–
Williams test compares mean angles of two or more samples by
rst transforming the proportions of owering individuals to ra-
dians; this test assumes a Von Mises distribution. e strength of
phenological isolation was evaluated using the 4S2 equation from
Sobel and Chen (2014; Appendix S1). Total reproductive isolation
was calculated using the average of the 2 years when strength values
diered by less than 15%. In three cases, yearly values diered by
more than 30%; thus, we present the individual values for each year
separately.
Pre‐pollination RI: oral traits
Floral morphology and color—We evaluated dierences in o-
ral morphology between A. flava, A. patens, and A. antirrhina by
measuring the following traits on 30 individuals of each species: (1)
corolla length, (2) total ower length including corolla and spur,
(3) corolla mouth height, (4) corolla mouth
width, (5) petal are, (6) anther height, (7)
stigma height, and (8) corolla constriction.
Herkogamy was calculated as the dierence
between anther and stigma height. Since color
dierences were large, corolla color was as-
sessed qualitatively. To determine the degree
of morphological overlap between the study
species, rst we conducted a principal com-
ponent analysis (PCA) in R with the stats
package (R Core Team, 2018). A multivariate
analysis of variance (MANOVA) was used to
compare oral phenotypes with traits 1–5 and
8 as dependent variables and species as the
main factor. Traits 6 and 7 were not included
because Achimenes species are protandrous
and the length of stamens and style vary with
time.
Nectar production and sugar concentra-
tion—In the greenhouse, we tagged six ower
buds on 13 plants of each Achimenes species.
We extracted nectar with 1‐μL micropipettes
through a small hole perforated at the base
of the corolla. e length of the nectar col-
umn was measured with an analog caliper
(DialMax, Willi Hahn Co., Monticello, MN,
FIGURE 1. Reproductive phenology of three sympatric species of Achimenes (Gesneriaceae)
monitored at La Tzararacua, Michoacán, Mexico during 2014 and 2015.
1024 • American Journal of Botany
USA). Sugar concentration was measured with a hand ATC refrac-
tometer with temperature calibration and a range of 0–32% °Brix
(equivalent to the number of grams of solute per grams of solution).
We also analyzed the composition and quantity of nectar sugars us-
ing gas chromatography. Nectar was collected in lter paper and
eluted in water for posterior gas chromatographic and mass spec-
trometric analyses as described in Appendix S3. Nectar was mea-
sured from 4‐d‐old owers, when stigmas were clearly open and
turgid.
We used generalized linear models (GLMs) in the stats package
of R (R Core Team, 2018) to test for dierences between species
(predictor variable) in mean nectar volume per ower (response
variable; gamma distribution and inverse link function). We also
tested for dierences in sugar concentration between species, with
dierent error distributions depending on the nature of data (to-
tal concentration: gamma with inverse link; sucrose and glucose:
Gaussian with identity link; fructose: inverse Gaussian with 1/mu2
link; inositol: Poisson with log link; °Brix concentration: quasibino-
mial with log link). Analyses were performed with the stats package
in R (R Core Team, 2018).
Floral scents—Floral volatiles were quantied in the laboratory
infour individuals per species, which had been collected in the eld
and kept in a live collection in the greenhouses of ENES‐Morelia,
Universidad Nacional Autónoma de México. Volatiles were ex-
tracted from owers that were enclosed in glass jars by vacuuming
air for 7 h (from 08:00 to 15:00 hours) and eluted in hexane. Aer
elution, volatiles were analyzed with gas chromatography. Detailed
methods are described in Appendix S3.
Pre‐pollination RI: Pollinator visitation
Floral visitors at focal plants or plant patches of each species were
directly observed and recorded with video cameras (SONY, Japan,
and Panasonic, Japan) for 1‐h periods throughout the day (08:00–
16:00 hours) in sunny to cloudy‐misty weather,during the ower-
ing seasons of 2013–2015. Total observation times and sample sizes
are provided with pollinator visitation results. We recorded time of
visitation, identity and behavior of the visitor, and number of ow-
ers probed. Visitors that contacted the reproductive organs of the
owers were considered legitimate pollinators. We calculated polli-
nator visitation rates by pollinator functional group (i.e., humming-
bird, large bee, small bee, buttery) as the mean number of visits
per ower per hour. Since visitation rates were low and collection of
all oral visitors was not possible, we identied most pollinators to
the lowest possible taxonomic category from video recordings and
photographs. We searched the literature for information on length
of mouthparts (i.e., proboscis or beak) of the dierent pollinator
groups to assess their association with corolla length. Reproductive
isolation was calculated using RI4A equation of Sobel and Chen
(2014; Appendix S1).
Pre‐pollination RI: autonomous self‐pollination
We conducted a greenhouse experiment in 2015 to evaluate the
role of self‐pollination as a potential RI mechanism. We marked
three ower buds per plant on 24 individuals of each species and
assigned them to one of the following treatments: (1) autonomous
pollination (unmanipulated bagged owers), (2) hand cross‐polli-
nation (emasculated owers; pollen from two donors of the same
species, i.e., intraspecic crosses), and (3) hand self‐pollination
(pollen from owers of the same plant). We previously assessed
time of stigma receptivity by recording peroxidase activity each day
of anthesis (Kearns and Inouye, 1993), which corresponded to full
extension of stigma lobes in all species. Mixtures of pollen from
four fresh stamens were placed directly onto stigmas, ensuring
stigma surfaces were saturated with pollen. To compare the fruit
set of autonomous self‐pollination with the fruit set achieved un-
der natural conditions, in the eld we quantied the fruit set from
unmanipulated owers (one per plant in 30 plants per species). For
all treatments, we followed fruit development until maturity and
collected drymature capsulesprior to dehiscence.
We used generalized linear mixed models (GLMMs) in the lme4
package in R program (Bates etal., 2015; R Core Team, 2018) to
determine the eect of treatment on fruit set (binomial, logit link
function). Individual was included as a random factor. Contrasts
between treatments were performed with glht function and single‐
step method in package multcomp (Hothorn etal., 2008).
Post‐pollination RI: hybrid viability
In the greenhouse, we conducted intraspecic and interspecic
crosses in 2016 and 2017 to evaluate post‐pollination barriers to
hybrid fruit and seed production and potential costs of hybrid-
ization. We tagged 33 individual plants of A. antirrhina, 29 of
A. flava, and 52 of A. patens; however, nal sample sizes varied
from loss of ower buds or individuals during the study. ree
ower buds per individual were assigned to a dierent hand‐
pollination treatment, where each plant served as a pollen donor
and a pollen recipient for each of two congeners. Pollen was
saturated onto stigmas using anthers of the paternal species.
Approximately 2 months aer pollination, we counted and col-
lected dry mature capsules. Capsules were immediately placed in
petri dishes until they released the seeds. We took photographs
of all seeds produced by each fruit with a Stemi 350 stereoscope
and an Axiocam 105‐color (Carl Zeiss, Germany) and counted
viable and nonviable seeds. Preliminary work indicated that
aborted seeds have contorted shapes and smaller sizes; therefore,
we assessed seed shape and size to estimate the total number of
viable seeds produced per species (hereaer referred to as seed
production). For analyses, we used fruit set (fruits/owers) and
seed production as proxies of hybrid viability.
Aer inspecting residuals under a linear model, we used
GLMMs in the lme4 package (Bates etal., 2015; R Core Team,
2018) to test for the eect of treatment (intraspecic and both in-
terspecic crosses) on fruit set (binomial distribution, logit link
function) and seed production (Poisson distribution, log link
function). Individual plant was included as a random factor. Ad
hoc comparisons of the intraspecic pollination treatment vs.
each interspecic cross were computed with a two‐tailed test, us-
ing multcomp with single‐step method and multiple comparisons
package in R (Hothorn etal., 2008; R Core Team, 2018). Data for
2016 and 2017 were pooled because sample sizes in 2016 were
small (less than 13) and fruit set values were similar in both years.
For calculations of reproductive isolation at the level of fruit set
and seed production, we used the RI4A equation from Sobel and
Chen (2014), which considers the probability of gene ow between
species pairs (Appendix S1). RI values range from 1 (complete iso-
lation) to −1 (complete disassortative mating; RI = 0 indicates no
isolation; Sobel and Chen, 2014).
July 2019, Volume 106 • Ramírez‐Aguirre etal.—Avoidance of interspecific reproduction in Achimenes species • 1025
Total reproductive isolation
To understand the contributions of each reproductive barrier (eco-
geographic isolation, phenological isolation, oral isolation, and
hybrid fruit set and seed production) to total reproductive isola-
tion, we used the RI4E equation and calculations proposed by Sobel
and Chen (2014). Detailed methods are presented in Appendix
S1. We show the individual strengths of each barrier and the ab-
solute contributions of each barrier to total isolation excluding
ecogeographic isolation (to obtain RI estimates under sympatric
conditions).
RESULTS
Pre‐pollination RI: ecogeographic barriers
e three study species were found in both sympatric and allopatric
populations. Ecogeographic isolation values between species pairs
were over 0.6 in all cases (Table1). e more ecogeographically iso-
lated species pair was A. patens and A. antirrhina (0.74) and the
least isolated A. antirrhina and A. flava (0.64).
Pre‐pollination RI: owering phenology
e owering times of the three study species overlapped during
August and September, but initial owering dates and peaks dif-
fered (Fig.1). ere were no signicant dierences in mean ow-
ering times between species in 2014 (Watson–Williams test F2, 18 =
0.03, P = 0.970), nor in 2015 (F2, 21 = 0.57, P = 0.574). Mean ower-
ing times corresponded to the rst 2 weeks of August in 2014 and
mid August in 2015. RI due to owering phenology varied between
species pairs and was higher in 2014 than in 2015 (RI range, 2014:
0.43–0.94, 2015: 0.09–0.70; Table1).
Pre‐pollination RI: oral traits
Floral morphology—Floral phenotypes diered considerably be-
tween species and showed no overlap in morphological space
(MANOVA, F12, 160 = 170.5, P = 2.2 × 10−16; Fig. 2; Appendix S4).
e traits that contributed most to variation were corolla length,
total ower length, and corolla mouth length for PC axis 1 (56.4%
of total variance) and corolla mouth width and petal are to PC
TABLE 1. Strength of reproduction isolation for individual barriers and total isolation values between three Achimenes species from Central Mexico. Absolute
contributions of each barrier to total isolation include all barriers (AC) and barriers acting in sympatry (AC‐Sym). Relative contributions to total isolation(not shown)
equaled ACs, except for theA. patens × A. avacross.
Maternal × Paternal A. antirrhina × A. ava A. antirrhina × A. patens
Barrier Strength AC AC‐Sym Strength AC AC‐Sym
Ecogeographic 0.638 0.64 — 0.743 0.74
Phenologicala 0.830 0.30 0.83 0.460, 0.149 0.12, 0.04 0.46, 0.15
Floral 1 0.06 0.17 1 0.14, 0.22 0.54, 0.85
Fruit set 0.660 0 0 0.560 0 0
Total 1 1 1 1
A. ava × A. antirrhina A. ava × A. patens
Ecogeographic 0.638 0.64 — 0.716 0.72
Phenologicala 0.923, 0.493 0.33, 0.18 0.92, 0.49 0.432 0.12 0.43
Floral 1 0.03, 0.18 0.08, 0.51 1 0.16 0.57
Fruit set 0.700 0 0 −0.006 0 0
Seed production 0 0 0 0.35 0 0
Total 1 1 1 1
A. patens × A. antirrhina A. patens × A. ava
Ecogeographic 0.743 0.74 /0.74 — 0.715 0.72 —
Phenologicala 0.429, 0.093 0.11, 0.02 0.43, 0.09 0.494 0.14 0.49
Floral 1 0.15, 0.24 0.57, 0.91 0.800 0.11 0.40
Fruit set 0.210 0 0 0.089 0.01 0.02
Seed production 0.500 0 0 0.292 0.01 0.04
Total 1 1 0.99 0.95
aMean values for 2014 and 2015 phenological reproductive isolation were used for yearly strength values that differed by less than 15%. Individual values for each year are given and
separated by a comma when differences exceeded 30.
FIGURE 2. Principal component analysis of oral traits for three sym-
patric species of Achimenes from La Tzararacua, Michoacán, Mexico.
Achimenes antirrhina (A), A. ava (B), A. patens (C).
A
BC
1026 • American Journal of Botany
axis 2 (28.2% of total variance). Corolla lengths coincided with
the mouthparts of the main pollinators of each Achimenes species
(Table2). Corolla color also diered between species (Fig.2).
Nectar production and sugar composition—Nectar volume
diered among the three Achimenes species (χ2 = 46.3, df = 2,
P= 8.9 × 10‐11; Table3); the highest volume was for ornithophi-
lous A. antirrhina (6.6 ± 47.62 μL), and the lowest was for A. flava
(0.8 ± 5.59 μL). Sucrose was the most abundant nectar sugar in all
three species, ranging between 107 and 135 μg/μL (Table 3) and
did not dier in concentration among Achimenes species (χ2 = 1.4,
df=2, P = 0.503). Hexoses (fructose and glucose) and inositol were
three orders of magnitude lower than sucrose; fructose and glucose
concentration diered among species (χ2 = 7.1, df = 2, P = 0.028;
χ2=6.0, df = 2, P = 0.049, respectively), while inositol concentration
did not (χ2=0.17, df = 2, P = 0.917; Table3). Total sugar concentra-
tion ranged between 108 and 159 μg/μL and did not dier among
species (χ2 = 1.3, df = 2, P = 0.517; Table3), neither did sugar con-
centration expressed as °Brix (χ2 = 2.6, df = 2, P = 0.276).
Floral scents—Organic volatiles in owers included the terpenoids
pinene, limonene, cineole, the benzenoid naphthalene, and the
fatty‐acid derived alkene tetradecane, but the presence and con-
centration of these compounds varied among species (Table4). e
oral scent prole of A. antirrhina showed two of the ve volatiles,
while the prole of A. flava and A. patens showed four of the ve
volatile compounds. e last two species diered in one volatile
compound; limonene was exclusive to A. patens, and cineole was
exclusive to A. flava (Table4).
Pre‐pollination RI: pollinator visitation
e main pollinators of the study species were hummingbirds for
A. antirrhina, bees for A. flava, and butteries for A. patens (Table5).
Amazilia beryllina was the only hummingbird species that visited
A. antirrhina, and it was not observed at any other plant species at
the study site. is hummingbird generally probed various ow-
ers within a patch, but territorial behavior was not observed. Bee
visitors to owers of A. flava included medium‐sized bees (tribes
Centridini and Eucerini), small halictid bees and one crabronid
wasp species; a buttery from the genus Pieris visited owers once
in 2013. All lepidopterans observed at A. patens were in the families
Hesperiidae and Pieridae. rips and the Crabronidae wasp were
also observed visiting A. patens. rips acted as nectar robbers be-
cause they did not contact the reproductive organs of the owers,
while Crabronid wasps may be occasional pollinators. Reproductive
isolation through pollinator visitation was complete for most spe-
cies pairs except for A. patens–A. flava (Table1).
Pre‐pollination RI: autonomous self‐pollination
All three species are self‐compatible since they produce fruit and seed
aer hand‐self pollination (Fig.3). However, the values of autono-
mous fruit set ranged between 0 and 4%, while hand‐cross pollina-
tion consistently resulted in fruit set higher than 50% (Fig.3); thus,
autonomous self‐pollination does not contribute to RI. Fruit set from
hand cross‐ and self‐pollination were higher than fruit set from nat-
ural pollination (A. antirrhina: χ2 = 5.9, df = 2, P = 0.049; A. flava:
χ2=60.0, df = 3, P < 0.0001; A. patens: χ2 = 57.1, df = 3, P < 0.0001).
TABLE 2. Mean (±SEM) corolla length and corolla mouth width of three sympatric Achimenes species from Central Mexico, and bill or proboscis length (range)of their
primary pollinators recorded from the literature.
Species N
Corolla length
(mm)
Corolla width
(mm) Main pollinator
Bill/ proboscis length
(mm) Source
A. antirrhina 30 27.3 ± 0.70 6.13 ± 0.14 Amazilia beryllina 18-21 Howell, 2003
A. flava 30 10.1 ± 0.15 5.72 ± 0.17 Centris aff. atripes 11-14a Roubik et al., 1995
A. patens 30 14.5 ± 0.25 3.20 ± 0.13 Urbanus sp. 16-17 Bauder et al., 2015
aProboscis lengths of other Centris species collected in Mexico.
TABLE 3. Mean (±SEM) oral nectar volume and sugar concentration for three species of Achimenes from Central Mexico. Nectar samples were obtained from
greenhouse plants previously collected at La Tzararacua, Michoacán.
Species NNectar vol. (μL) Fructose (μg/μL) Glucose (μg/μL) Sucrose (μg/μL) Inositol (μg/μL) S/H (μg/μL) Total (μg/μL) °Brix (%)
A. antirrhina 11 6.6 ± 1.04a0.5 ± 0.64a 0.02 ± 0.014b 136 ± 28.7a0.2 ± 0.11a805 ± 328.5 136 ± 31.3a33 ± 3.8a
A. flava 10 0.8 ± 0.11b0.2 ± 0.32b 0.07 ± 0.015a 107 ± 32.7a0.2 ± 0.15a621 ± 216 108 ± 27.0a40 ± 3.2a
A. patens 13 3.2 ± 0.4c0.2 ± 0.30b 0.03 ± 0.014b 158 ± 28.7a0.2 ± 0.13a835 ± 189.7 159 ± 29.46a33 ± 3.7a
Notes: Nectar volume (vol.), raw values for fructose and sugar concentration are given for easier comparison. S/H, proportion of sucrose (S) to common hexoses (H = fructose + glucose);
°Brix, sugar concentration (expresses mass/mass relation). Different letters within a column indicate a difference between species.
TABLE 4. Mean relative percentage (±SEM) of oral scent compounds of three Achimenes species collected at La Tzararacua site and grown in a greenhouse.
Compound KRI A. antirrhina A. ava A. patens
α-Pinene 939 58.0 ± 1.78 52.4 ± 17.30 38.5 ± 20.91
Limonene 1029 — — 13.6 ± 7.88
1,8-Cineole 1031 — 5.0 ± 5.03 —
cis-Decahydronaphthalene 1099 41.9 ± 1.78 20.2 ± 9.31 28.8 ± 18.99
Tetradecane 1400 — 22.3 ± 13.71 17.0 ± 9.82
Note: KRI, Kovats retention index.
July 2019, Volume 106 • Ramírez‐Aguirre etal.—Avoidance of interspecific reproduction in Achimenes species • 1027
Post‐pollination RI: hybrid viability
Interspecic crosses produced fruits in eight of nine parental com-
binations; however, fruit set was asymmetric between crosses ac-
cording to the identity of the donor and recipient species (Figs.4,5).
Fruit set ranged between 53 and 75% for intraspecic crosses and
between 11 and 76% for interspecic crosses (Fig.4). When A. an-
tirrhina was the pollen recipient, hybrid fruit set was lower than in-
traspecic fruit set in both interspecic crosses (χ2 = 10.5, df = 2, P =
0.005). When A. flava was the pollen recipient, hybrid fruit set was
signicantly lower than intraspecic fruit set only when crossed
with A. antirrhina (χ2 = 21.8, df = 2, P < 0.0001). When A. patens
was the pollen recipient, hybrid fruit set values were lower, but they
did not signicantly dier from intraspecic fruit set (χ2 = 5.2,
df = 2, P = 0.075). Reproductive isolation estimates for interspecic
crosses ranged from −0.006 for the cross A. flava × A. patens to 0.70
for the A. ava × A. antirrhina cross (Table1).
Seed production varied with the identity of the pollen recipient
and donor species. Seed production ranged between 304 and 946
for intraspecic crosses and between 146 and 311 for hybrid crosses,
andthe cross A. flava × A. antirrhina did not yield any viable seed
(Fig. 5). For both A. flava and A patens as pollen recipients, hybrid
seed production values were signicantly lower than intraspecic
seed production (χ2 = 367.8, df = 1, P < 0.0001; χ2 = 3075.3, df = 2,
P< 0.0001, respectively). RI values ranged from 0.29 (A. patens ×
A. flava) to 0.5 (A. patens × A.antirrhina). Crosses with A. antirrhina
as a maternal plant were not assessed due to high fruit mortality.
DISCUSSION
Pre‐ and post‐mating mechanisms of
reproductive isolation
is study evaluated dierent plant traits that
might be involved in reproductive isolation
among three congeneric sympatric Gesneriaceae
species. Achimenes flava, A. antirrhina, and
A. patens are habitat specialists that occur on hu-
mid rocky slopes of river canyons in seasonal en-
vironments in the mountains of central Mexico;
thus, we predicted they would oen coexist in
such microhabitats. However, the results showed
that ecogeographic isolation is an important
pre‐pollination barrier when estimated from
relatively large geographic scales (1‐km2 reso-
lution layers), indicating that sympatric assem-
blages are not as common as expected and that
sympatric sites should be hotspots of selection
for other pre‐pollination mechanisms of RI.
Flowering phenology largely overlapped
between species at a site of sympatry; however,
TABLE 5. Mean pollinator visitation rates (±SEM) of three Achimenes species at La Tzararacua, Michoacán, Mexico during 2013–2015, calculated as mean number of
visits ower −1 h−1. N is the total number of observation hours.
Species Pollinator taxon
Mean number of visits ower−1 h−1
2013 (N = 12) 2014 (N = 10) 2015 (N = 5, 6, 8)a
A. antirrhina Trochilidae 0.10 ± 0.011 0.19 ± 0.012 0.6 ± 0.4
Amazillia beryllina
A. flava Hymenoptera 0.14 ± 0.032 0.23 ± 0.091 0.16 ± 0.17
Centris aff. atripes (Apidae)
Eucerini sp.
Halictidae (1 sp.)
Crabronidae (1 sp.)
Pieris sp.
A. patens Lepidoptera 0.34 ± 0.140 0.12 ± 0.07 0.5 ± 0.26
Urbanus dorantes, U. proteus
(Hesperiidae)
Pieridae (aff. Pieris sp.)
Hymenoptera 0.25 ± 0.25
Crabronidae (1 sp.)
aSample sizes, respectively, for A. antirrhina, A. flava, A. patens.
FIGURE 3. Mean fruit set (±SEM) from four pollination treatments used to determine the capac-
ity for autonomous self‐pollination in three sympatric species of Achimenes from Central Mexico.
Hand pollinations were done in the greenhouse and natural pollination was quantied at La
Tzararacua, Michoacán, during 2015. Letters indicate signicant dierences between treatments
within a species.
1028 • American Journal of Botany
owering peaks diered signicantly, particularly in 2014. In most
cases, owering phenology was an important contributor to RI, but
it varied between years and species pairs. e overlapping owering
seasons of the study species may be associated with their pseudo‐
annual life history, with aboveground stems that die and regrow ev-
ery year from underground rhizomes at the beginning of the rainy
season. is life cycle restricts the time available for growth and
reproduction, as has been described for other
geophytic plant species (Dafni etal., 1981).
Hence, if closely related sympatric species are
constrained to ower during the same period
of the year, selection may favor oral traits
that promote specialization in pollination
systems (Rathcke and Lacey, 1985).
Floral isolation is, according to our results,
the most important reproductive barrier
among the study species. Only twice during
the study, the same pollinator was observed
visiting owers of two Achimenes species,
which suggests that particular oral traits at-
tract certain kinds of oral visitors and deter
others (see discussion below). Floral isolation
through morphology (mechanical isolation)
is one of the most important RI mechanism
among sympatric species in various plant
genera (e.g., Asclepias, Kephart and eiss,
2003; Costus, Kay, 2006; Spiranthes sinensis
complex, Tao et al., 2018), but ethological
isolation through signals and rewards that in-
uence pollinator behavior are also important
(Schemske and Bradshaw, 1999; Klahre etal.,
2011; Byers etal., 2014).
Autogamy may act as a reproductive
barrier in some species (Levin, 1971; Brys
etal., 2016), but it is unlikely to be relevant
in Achimenes, given the low fruit set re-
sulting from autonomous self‐pollination.
Furthermore, post‐zygotic barriers were
weak in most species pairs, indicating that
reproductive isolation is incomplete at the
level of hybrid viability, a nding that has
been reported for other Gesneriaceae spe-
cies (Johnson etal., 2015; Zhang etal., 2017).
Fruit and seed production from interspecic
crosses varied according to the identity of the
pollen recipient species, and they were oen
asymmetrical (Fig. 3). ese results may re-
ect the inability of pollen tubes from smaller
owers (i.e., A. flava, A. patens) to develop
past their autotrophic phase down to the ova-
ries of the long‐styled A. antirrhina, but this
idea needs to be tested. Other mechanisms
that explain asymmetries in hybrid viability
in other species are genetic and intracellular
incompatibilities, pollen–pistil interactions,
triploid endosperm interactions and/or ma-
ternal eects (Turelli and Moyle, 2007). Our
results of hybrid viability, estimated from fruit
and seed production are congruent with tests
of hybridization, where several Gesneriaceae
species have a high potential to produce hybrid progeny; nonethe-
less, pollen viability may be reduced in some Achimenes hybrids, in-
dicating barriers may be acting at the level of hybrid fertility (Cooke
and Lee, 1966; Wiehler, 1983).
Extrinsic mechanisms that act on individual hybrids, such as low
competitiveness in the parental environment might also account for
the low occurrence of hybrid phenotypes in the wild (Widmer etal.,
FIGURE 4. Mean fruit set (±SEM) obtained from intra‐ and interspecic crosses between three
Achimenes species from Central Mexico. Letters indicate species and shapes indicate maternal
species for each cross: A /diamond= A. anthirrhina, F /triangle = A. ava, P /circle = A. patens.
Letters over symbols indicate signicant dierences between treatments within each maternal
species.
FIGURE 5. Mean seed production per fruit (±SEM) obtained from intra‐ and interspecic crosses
between three Achimenes species from Central Mexico. Letters indicate species and shapes indi-
cate maternal species for each cross: A /diamond= A. anthirrhina, F /triangle= A. ava, P /circle=
A. patens.Letters over symbols indicate signicant dierences between treatments within each
maternal species.
July 2019, Volume 106 • Ramírez‐Aguirre etal.—Avoidance of interspecific reproduction in Achimenes species • 1029
2009). We registered two individuals with oral phenotypes that
were intermediate between A. flava and A. patens in 2013, but we
did not nd them the following years. Interestingly, pollen viability
in hybrids from A. flava and A. patens is less than 30% (Wiehler,
1983), suggesting that even if hybrid fruits are produced, hybrid in-
dividuals are possibly poor pollen donors. Overall, ndings from
this and other studies suggest that there is a costinvolved in the
production of hybrid progeny.
Floral features that contribute to reproductive isolation—Dier-
ent oral morphologies in closely related species may promote in-
traspecic pollination by placing pollen dierentially on the bodies
of particular pollinators or by attracting specic functional groups
of pollinators (e.g., Pauw, 2006; Martén‐Rodríguez et al., 2009).
Two of the three study species, A. antirrhina and A. patens, have
oral morphologies that promote eective pollen transfer by a sin-
gle functional group of pollinators and restrict access to unwanted
visitors (Fig.2). Narrow, long corollas and spurs in A. patens only
allow access to nectar to insects with relatively long proboscides,
such as the observed hesperid butteries (see Table2). A similar
association has been described for various plant groups pollinated
by lepidopterans or long‐tongued ies (e.g., Whittall and Hodges,
2007; Pauw etal., 2009). Likewise, in the case of Achimenes antir-
rhina, corollas are tubular and only a few millimeters longer than
the mouthparts of their hummingbird pollinators (Amazilia beryl-
lina bill length: 19–21 mm; Howell, 2003). is type of mechani-
cal isolation has been described for various plant taxa, such as the
genera Aquilegia, Penstemon, and Costus (e.g., Fulton and Hodges,
1999; Castellanos etal., 2004; Kay, 2006), although the owers of
hummingbird‐pollinated plants are oen wide enough to be visited
by bees and other small insects. In the case of A. antirrhina, the
absence of bees might be associated with reduced attraction due
to ower color; however, an assessment of ower color spectra and
color vision of local bees would be necessary to test this idea.
e manipulation of pollinator behavior based on pollinator
senses, feeding preferences and energetic demands may contribute
to preventing heterospecic visitation (i.e., ethological isolation,
Grant, 1994). For example, dierences in pollinator attraction medi-
ated by the quantity or quality of oral rewards may play an import-
ant role at preventing interspecic pollen transfer (Mitchell, 1993;
Schemske and Bradshaw, 1999). In this study, the nectar volume
produced per ower diered between the three Achimenes species
consistent with previous studies (Baker and Baker, 1983; Cruden
et al., 1983), higher volumes were produced by hummingbird‐
pollinated A. antirrhina and the lowest volumes by bee‐pollinated
A. flava. Nectar sugar composition has also been shown to dier
according to the preferences of particular pollinators (Baker and
Baker, 1983). However, in the present study, nectar composition did
not contribute to oral isolation because sucrose was the dominant
sugar in the nectar of all three Achimenes species and the content
of other sugars varied little among species. ese results suggest
that nectar sugar composition is phylogenetically conserved in
Achimenes, similar to the case of hummingbird‐ and bee‐pollinated
species of the Brazilian clade Sinningiae (Gesneriaceae; Perret etal.,
2001).
In the case of oral scents, three volatiles were shared between
the insect‐pollinated species, and two of these were present in
hummingbird‐pollinated A. antirrhina. e remaining volatiles in-
cluded one compound that was exclusive to bee‐pollinated A. flava
(1,8‐cineole) and one exclusive to buttery‐pollinated A. patens
(limonene). e nding of only two oral volatiles in A. antirrhina
agrees with ndings for other ornithophilous species and suggests
that nearly scentless owers evolve in lineages pollinated by hum-
mingbirds (Knudsen etal., 2004), although sense of smell has been
little studied in these vertebrates (e.g., Goldsmith and Goldsmith,
1982). In contrast, visual cues and oral scents are common in bee‐
pollinated species (Dobson, 2006). Interestingly, of the four scents
in A. flava, 1,8‐cineole and α‐pinene are commonly found in ow-
ers pollinated by male euglossine bees, and tetradecane is common
in owers pollinated by nectar‐seeking bees (Gerlach and Schill,
1991; Dobson, 2006; Martel et al. 2019). In contrast, terpenoids
such as 1,8‐cineole and limonene are apparently not well perceived
by butteries (Andersson and Dobson, 2003); thus, the function
of limonene in owers of A. patens needs to be further explored.
Finally, the unexpected presence of naphthalene in all species may
be an herbivore deterrent, as suggested for Magnolia (Azuma etal.,
1996). Future studies should address the association between vol-
atiles and pollination systems to determine their potential role in
reproductive isolation in the Gesneriaceae.
CONCLUSIONS
Our results highlight the importance of pollination system spe-
cialization at preventing interspecic pollination and avoiding the
costs of hybrid progeny production, allowing the co‐occurrence of
closely related species with reduced reproductive interference. We
determined that mechanical (oral morphology) and ethological
traits (nectar volume and oral volatiles) contribute to promote
intraspecic visitation by particular pollinators. In contrast, phe-
nology does not allow full temporal separation of reproductive sea-
sons and post‐zygotic barriers are weak, generating conditions that
might favor pollinator‐mediated selection on oral traits to reduce
reproductive interference. ese results suggest a potential role of
reinforcement (i.e., selection that acts on particular traits to reduce
the costs associated with the production of hybrids of low viability
or performance; Hopkins, 2013) in the oral diversication of the
genus Achimenes. Future research investigating the role of phyloge-
netic constraints on phenological and nectar traits and the role of
reinforcement in the diversication of oral traits in tropical geo-
phytes is warranted.
ACKNOWLEDGEMENTS
We thank Andrea Padilla, Germán Avila‐Sakar for help with
eldwork; P. Emiliano Cortés, Violeta Patiño, and Gumersindo
Sánchez‐Montoya for technical assistance; and two anonymous
reviewers for insightful comments to earlier versions of this man-
uscript. We dedicate this article to the memory of our friend and
colleague Constantino Orduña, who initiated the ex situ collection
of Achimenes used in this study. Posgrado en Ciencias Biológicas,
Universidad Nacional Autónoma de México provided funding and
support for the development of the doctoral dissertation project of
Erandi Ramírez‐Aguirre. is project was funded by grants from
Universidad Nacional Autónoma de México (Dirección General
de Personal Académico PAPIIT IA208416, IA207618, IV200418;
Programa de Becas Posdoctorales en la UNAM), Programa
Iberoamericana de Ciencia y Tecnología para el Desarrollo RED
CYTED‐SEPODI (417RT0527), and CONACyT‐Mexico (Projects
1030 • American Journal of Botany
Laboratorio Nacional de Análisis y Síntesis Ecológica 271449,
280505, 293701, 299033 to M.Q. and S.M.R.; Proyecto Repositorio
Institucional 271432 and C.B. 155016 to S.M.R., and CVU/scholar-
ship 413896/261462 to E.R.A.).
AUTHOR CONTRIBUTIONS
E.R.A. and S.M.R. designed the study, collected and analyzed data,
and wrote the manuscript. Y.M.D., F.J.E.G. and G.Q.A. analyzed
nectar and oral scent data; G.Q.A. and Y.M.D. conducted inter-
specic pollinations; and M.Q. and K.O. contributed to eldwork
and manuscript writing.
DATA ACCESSIBILITY
Calculations of reproductive isolation were based on supporting
information from Sobel and Chen (2014) at https ://onlin elibr ary.
wiley.com/doi/full/10.1111/evo.12362 . Species presence data and
geographic information were accessed from www.gbif.org; http://
www.world clim.org/version1; http://www.conab io.gob.mx/infor m
acio n/gis/; and https ://web.archi ve.org/web/20170 51922 1949/
http://world grids.org/doku.php/wiki:layer s#dem-deriv ed_para
m eters .
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
supporting information tab for this article.
APPENDIX S1. Reproductive isolation equations from Sobel and
Chen (2014) used in the analysis of reproductive barriers among
three Achimenes species from Central Mexico.
APPENDIX S2. Methods used to estimate ecogeographic isolation
between three Achimenes species from Central Mexico.
APPENDIX S3. Methods for nectar and scent analyses used for
three Achimenes species from Central Mexico.
APPENDIX S4. Floral trait measurements of three Achimenes spe-
cies from La Tzararacua, Michoacán, México.
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