Selfing in epiphytic bromeliads compensates for the limited pollination services provided by nectarivorous bats in a neotropical montane forest

Abstract

Plants with specialized pollination systems frequently exhibit adaptations for self-pollination, and this contradictory situation has been explained in terms of the reproductive assurance function of selfing. In the Neotropics, several plant lineages rely on specialized vertebrate pollinators for sexual reproduction, including the highly diverse Bromeliaceae family, which also displays a propensity for selfing. Thus far, the scarce evidence on the role of selfing in bromeliads and in other neotropical plant groups is inconclusive. To provide insights into the evolution and persistence of self-fertilization in the breeding systems of Bromeliaceae, we studied four sympatric epiphytic species from the genus Werauhia (Tillandsioideae) in Costa Rica. We documented their floral biology, pollination ecology, and breeding systems. We estimated the contribution of selfing by comparing the reproductive success between emasculated flowers requiring pollinator visits and unmanipulated flowers capable of selfing and exposed to open pollination across two flowering seasons. The studied species displayed specialized pollination by nectar-feeding bats as well as a high selfing ability (autofertility index values > 0.53), which was attained by a delayed selfing mechanism. Fruit set from natural cross-pollination was low (<26% in both years) and suggested limited pollinator visitation. In line with this, we found a very low bat visitation to flowers using video-camera recording, from 0 to 0.24 visits per plant per night. On the contrary, the contribution of selfing was comparatively significant since 54-80% of the fruit set from unmanipulated flowers can be attributed to autonomous self-pollination. We concluded that inadequate cross-pollination services diminished the reproductive success of the studied Werauhia, which was compensated for by a delayed selfing mechanism. The low negative effects of inbreeding on seed set and germination likely reinforce the persistence of selfing in this bromeliad group. These results suggest that selfing in bat-pollinated bromeliads may have evolved as a response to pollinator limitation.

Full text

AoB PLANTS, 2024, 16, plae011
https://doi.org/10.1093/aobpla/plae011
Advance access publication 26 February 2024
Studies
© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/),
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Received: 18 October 2023; Editorial decision: 1 February 2024; Accepted: 23 February 2024
Studies
Selfing in epiphytic bromeliads compensates for the
limited pollination services provided by nectarivorous bats
in a neotropical montane forest
Stephanie Núñez-Hidalgo1 and Alfredo Cascante-Marín*,2
1Sistema de Estudios de Posgrado, Universidad de Costa Rica, San Pedro de Montes de Oca, 11501-2060 San José, Costa Rica
2Escuela de Biología y Centro de Investigación en Biodiversidad y Ecología Terrestre (CIBET), Universidad de Costa Rica, San Pedro de Montes
de Oca, 11501-2060 San José, Costa Rica
*Corresponding author’s e-mail address: alfredo.cascante@ucr.ac.cr
Associate Editor: Gerardo Arceo-Gómez
Abstract. Plants with specialized pollination systems frequently exhibit adaptations for self-pollination, and this contradictory situation has been
explained in terms of the reproductive assurance function of selfing. In the neotropics, several plant lineages rely on specialized vertebrate pol-
linators for sexual reproduction, including the highly diverse Bromeliaceae family, which also displays a propensity for selfing. Thus far, the scarce
evidence on the role of selfing in bromeliads and in other neotropical plant groups is inconclusive. To provide insights into the evolution and
persistence of self-fertilization in the breeding systems of Bromeliaceae, we studied four sympatric epiphytic species from the genus Werauhia
(Tillandsioideae) in Costa Rica. We documented their floral biology, pollination ecology and breeding systems. We estimated the contribution of
selfing by comparing the reproductive success between emasculated flowers requiring pollinator visits and un-manipulated flowers capable of
selfing and exposed to open pollination across two flowering seasons. The studied species displayed specialized pollination by nectar-feeding
bats as well as a high selfing ability (auto-fertility index values > 0.53), which was attained by a delayed selfing mechanism. Fruit set from natural
cross-pollination was low (<26% in both years) and suggested limited pollinator visitation. In line with this, we found a very low bat visitation to
flowers using video-camera recording, from 0 to 0.24 visits per plant per night. On the contrary, the contribution of selfing was comparatively
significant since 54–80% of the fruit set from un-manipulated flowers can be attributed to autonomous self-pollination. We concluded that inad-
equate cross-pollination services diminished the reproductive success of the studied Werauhia, which was compensated for by a delayed selfing
mechanism. The low negative effects of inbreeding on seed set and germination likely reinforce the persistence of selfing in this bromeliad
group. These results suggest that selfing in bat-pollinated bromeliads may have evolved as a response to pollinator limitation.
Keywords: Breeding systems; Bromeliaceae; chiropterophily; Costa Rica; pollinator limitation; reproductive assurance.
Introduction
Selng or the ability to self-fertilize in plants, is a relatively
common reproductive strategy among angiosperms (~20%)
(Barrett, 2002), and in several species, the oral mechanisms
that facilitate selng also co-exist with specialized pollin-
ation systems in a mixed mating system (Fenster and Martén-
Rodríguez, 2007). The maintenance of selng as part of mixed
mating systems in plants is largely attributed to its benets
as a ‘reproductive assurance’ mechanism in the face of unre-
liable cross-pollination (Mallick, 2001; Herlihy and Eckert,
2002; Kalisz et al., 2004; Moeller and Geber, 2005; Moeller,
2006; Fenster and Martén-Rodríguez, 2007; Zhi-Quiang
and Quing-Jun, 2008; Martén-Rodríguez and Fenster, 2010;
Busch and Delph, 2012; Jones et al., 2013).
The ability to self-fertilize requires the loss of
self-incompatibility mechanisms and the existence of oral
biology adaptations to facilitate the autonomous deposition
of self-pollen onto the stigma. These mechanisms include the
absence of intra-oral herkogamy (Webb and Lloyd, 1986)
and dichogamy (Bertin and Newman, 1993). The establish-
ment of self-fertilization is also contingent on the absence
or reduced inbreeding depression effects (Charlesworth and
Charlesworth, 1987; Eckert et al., 2006). In addition, for
selng to provide reproductive assurance, the reproductive
success of a species must be constrained by pollen availability
or pollinator services (i.e. pollen limitation) (Eckert et al.,
2006), and it should not incur in pollen and ovules discount
(Knight et al., 2005).
The Bromeliaceae family is a very diverse group of mono-
cotyledonous plants, almost entirely restricted to the American
continent (Benzing, 2000). They contribute signicantly to the
oristic diversity of vascular epiphytic oras in the Neotropics
(Cascante-Marín and Nivia-Ruíz, 2013). Bromeliads possess
specialized pollination systems that involve vertebrate pol-
linators (hummingbirds and nectarivorous bats) and insects
to a lesser degree, mainly bees (Benzing, 2000; Kessler and
Krömer, 2000; Aguilar-Rodríguez et al., 2019a; Kessler et
al., 2020). Even though most bromeliads exhibit adaptations
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2AoB PLANTS, 2024, Vol. 16, No. 2
for cross-pollination, nearly two-thirds of the species inves-
tigated for their reproductive systems are capable of selng.
This is more frequent in the subfamilies Tillandsioideae and
Pitcairnioideae (Cascante-Marín and Núñez-Hidalgo, 2023).
However, little attention has been paid to detailed studies
of selng mechanisms and their adaptive value in neotrop-
ical plants as a whole. Previous works (Wendt et al., 2002;
Matallana et al., 2010) have proposed that the prevalence of
selng among bromeliads represents a reproductive isolation
strategy (sensu Levin, 1971) to minimize the negative effects
of hybridization in sympatry. Nevertheless, the evidence sup-
porting either hypothesis (‘reproductive assurance’ or ‘repro-
ductive isolation’) is inconclusive in this important group of
monocots (Cascante-Marín and Núñez-Hidalgo, 2023).
The mechanisms of selng vary with regard to the pre-
cise moment of its occurrence, and they dene the role of
selng, which has evolutionary consequences for plant tness
(Lloyd, 1992; Lloyd and Schoen, 1992; Brys and Jacquemyn,
2011). Selng may occur either before anthesis (prior
selng), during anthesis when the ower is exposed to cross-
pollination (competing selng), or at the end of its life (delayed
selng) (Schoen and Lloyd, 1992). Selng that occurs late in
the ower’s life (‘delayed selng’), when the possibility of
cross-pollination has passed, is likely to result in reproductive
assurance (Fenster and Martén-Rodríguez, 2007; Goodwillie
and Weber, 2018). Delayed selng does not interfere with
pollen pick-up by pollinators or with the stigma’s receipt of
crossed pollen, hence decreasing pollen and ovule discounting,
respectively (Lloyd, 1992; Herlihy and Eckert, 2002).
This study seeks to further our understanding of the sexual
reproductive systems of neotropical plants, particularly the
evolution and maintenance of selng in the Bromeliaceae
family. Using information from oral biology, pollination
ecology, and breeding systems of species from the genus
Werauhia J. R. Grant in the subfamily Tillandsioideae, we
intend to provide insights into the ecological causes for the
persistence and predominance of self-fertilization in this
plant group. Werauhia is proposed as a monophyletic group
(Barfuss et al., 2005) and is represented by one hundred rec-
ognized species (Gouda and Butcher, 2016 and cont. updated)
of epiphytic life-form and distributed mainly on the moun-
tains of southern Central America (Costa Rica and Panama)
(Grant, 1995; Morales, 2003). Previous studies in Werauhia
indicate the presence of specialized pollination systems
involving nocturnal nectarivorous bats (Aguilar-Rodríguez et
al., 2019a) and hummingbirds (Lasso and Ackerman, 2004),
as well as high selng ability in W. gladioliora (Cascante-
Marín et al., 2005; Tschapka and von Helversen, 2007), W.
nutans and W. noctiorens (Aguilar-Rodríguez et al., 2019b),
and W. sintenisii (Lasso and Ackerman, 2004).
We studied four Werauhia species that coexist simpatrically
in a Costa Rican montane forest and characterized their oral
biology (herkogamy, dichogamy, anthesis and senescence be-
haviour of owers), pollination system (identied the main
pollinators and their visitation rates) and the components of
their reproductive systems (i.e. self-compatibility, selng cap-
acity and presence of agamospermy). We also evaluated the
presence of inbreeding depression in self-fertilized progeny and
estimated the contribution of selng to reproductive success
in natural conditions during two owering episodes. We pre-
dict that our study species will exhibit high self-compatibility
and selng capacity, and if selng acts as a safeguard against
unpredictable cross-pollination (i.e. reproductive assurance),
then it should occur at the end of the ower life (‘delayed
selng’) (sensu Goodwillie and Weber, 2018).
Materials and Methods
Study site
This study was conducted at Cerros de (Hills of) La Carpintera
Protective Zone in Costa Rica, between 2018 and 2021. The
area comprises a small mountain formation in the eastern
region of the Central Valley of the country (9º52’–9º54” N;
83º57’–84º00’ W; 1500–1850 m asl). The site comprises 2396
hectares covered by patches of primary forest interspersed
with late secondary forest, and pastures (Sánchez et al., 2008).
The rainfall regime is seasonal, with a well-dened dry season
from December to April. The site has a rich epiphytic ora
and bromeliads are represented by 28 species from the genera
Aechmea (1 spp.), Catopsis (3), Guzmania (3), Pitcairnia (1),
Racinaea (2), Tillandsia (11), Vriesea (1), and Werauhia (6)
(Sánchez et al., 2008).
Study species
We selected the more abundant Werauhia species at the
study site: W. ampla, W. nephrolepis, W. pedicellata, and W.
subsecunda (Fig. 1). These are small to medium size and tank-
forming bromeliads that develop a single spiked or compound
inorescence per rosette. Werauhia species are distinguished
by having owers with nocturnal anthesis, zygomorphic
corollas with dull coloration (white or greenish), basal ap-
pendages of petals with the dactyloid divided apex, and
a cupular-shaped stigma without papillae (Grant, 1995).
The joint owering period of the four species extends from
November to August and shows signicant inter-specic tem-
poral displacement (Cascante-Marín et al., 2017). Voucher
specimens are deposited in the Luis Fournier O. Herbarium
(USJ) at the University of Costa Rica (W. ampla USJ-100246,
W. nephrolepis USJ-105232, W. pedicellata USJ-106525, and
W. subsecunda USJ-111865).
Floral biology
We documented nine oral traits for each species: (i) number
of owers per inorescence, (ii) oral display (number of
owers open per day), (iii) colour of the inorescence bracts
(peduncle, primary and oral bracts), (iv) corolla colour and
shape (campanulate or bilabiate), (v) stigma and anthers pos-
ition relative to the corolla mouth, (vi) stigma-anthers separ-
ation or herkogamy, (vii) anthesis time and ower longevity,
(viii) time of anther dehiscence and stigma receptivity or di-
chogamy, and (ix) mechanism of ower senescence. We tested
stigma receptivity with a peroxidase test (King, 1960; Kearns
and Inouye, 1993), using the presence of bubbling (observed
with a 20× hand magnifying glass) on the stigmatic surface as
an indicator of enzymatic activity.
We recorded the emission of oral volatile compounds
through an organoleptic test (i.e. smelling the open ower
and noticing any fragrance). Floral nectar volume and sugar
concentration were measured in owers from plants kept
in a shade house at the study site. Before anthesis, owers
were isolated to prevent nectar consumption by oral vis-
itors. Using glass capillary tubes, the accumulated volume
was measured 2–4 h after anthesis. A handheld refractometer
(Bellingham and Standley Ltd., UK) was used to estimate the
sugar concentration in Brix degrees.
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3Núñez-Hidalgo and Cascante-Marín – Selfing in epiphytic bromeliads compensates for the limited pollination
Floral visitors and visitation frequency
We recorded the ower visitors to each bromeliad species in
the forest with six video camera traps (Trophy cam, model
119476, Bushnell Corporation, Kansas, USA), during the
owering peaks of 2019, 2020, and 2021. The cameras were
set to record 15-second-long videos when activated, followed
by a period of 30 s of inactivity, during the day and night.
At each focal plant, the complete owering period of an in-
orescence was monitored. Only in a few cases, it was inter-
rupted due to battery depletion. The video analysis included:
(i) number of visits, (ii) visitor identity (e.g. bats, humming-
birds, others), (iii) time and duration of each visit, and (iv)
visitor behaviour (i.e. whether it contacted the anthers or
stigma). The visitation rate per night for the most frequent
visitors was determined by dividing the total number of re-
corded visits by the number of nights monitored each year.
To corroborate the chiropterophyllous transport of pollen,
we captured bats to examine if they were carrying pollen
from the studied species. We placed six mist nets (9 × 2.5 and
3 × 2.5 m) once or twice a week between January and February
2020, from 16:00 to 22:00 h, in sites considered as ‘passage
zones’ for bats (Wilson et al., 1996) and near owering in-
dividuals of the studied species. This sampling only included
the owering period of W. ampla and W. subsecunda. The
captured bats were identied following the taxonomic keys
of York et al. (2019). Pollen was obtained from the top of
the head and snout (cheeks-nose) using transparent adhesive
tape. The piece of tape with pollen was attached to a micro-
scope glass slide and a sampling area of 4.6 cm2 was visually
scanned under a light microscope in the laboratory. We used a
reference pollen collection from the study site to identify the
pollen grains carried by the bats.
Figure 1. Studied species of Werauhia (Bromeliaceae: Tillandsioideae) in a montane forest, Cerros La Carpintera, Costa Rica. (A–D) W. ampla, (E–H)
W. nephrolepis, (I–L) W. pedicellata, (M–P) W. subsecunda. (B, F, J, N) Night-vision images of bats visiting inflorescences of the studied species and
recorded with video camera traps. (C, G, K, O) Flowers in anthesis. (D, H. L, P) Senescent flowers whose corollas have lost turgor. Scale bars = 10 cm
(A, E, I, M) and 1.0 cm (C, G, K, O).
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4AoB PLANTS, 2024, Vol. 16, No. 2
Controlled pollination treatments and breeding
systems evaluation
We conducted controlled pollinations on 73 plants (17 W.
ampla, 15 W. nephrolepis, 16 W. pedicellata, and 25 W.
subsecunda) kept in a shade house at the study site, from
September 2018 to July 2019. We performed four pollin-
ation treatments: (i) manual self-pollination, (ii) manual
cross-pollination, (iii) pollinator exclusion (autonomous
selng), and (iv) emasculation (test of agamospermy). The
agamospermy test included stigma removal to avoid un-
noticed contamination, this treatment did not affect further
oral anthesis. Hand pollinations were conducted 1‒2 h after
anthesis and owers from all treatments were bagged until
their senescence. All treatments were performed on each plant
and randomly assigned to owers in the same inorescence.
Fruit development was monitored in a monthly basis and the
proportion of developed fruit in each treatment was calcu-
lated before fruit dehiscence.
The components of the reproductive systems were esti-
mated using the parameters described by (Cascante-Marín
and Núñez Hidalgo, 2023): (1) the self-compatibility
index: SCI = Pa/ Px (Lloyd and Schoen, 1992), the auto-
fertility index: AFI = Ps/ Px (Lloyd and Schoen, 1992), and
the agamospermy index: AGI = Pag/ Px (Riveros et al.,
1996). For all indices: Pa = proportion of fruits after hand
self-pollination, Pag = proportion of fruits after ower
emasculation, Ps = proportion of fruits from owers ex-
cluded from visitors and Px = proportion of fruits after
hand cross-pollination.
Reproductive success and inbreeding depression
We estimated the reproductive success per pollination treat-
ment as the mean number of seeds per fruit in a sample of
8–52 fruits per treatment and species. Potential effects of
inbreeding depression at the population level were tested by
comparing seed production and seed germination capacity
between manually self- and cross-pollination treatments. We
conducted a germination test using seeds from 8 to 46 fruits
per treatment (8–12 plants per species). Seeds were mixed
and a sample of 480 seeds per treatment was distributed
among 12 replicates of 40 seeds placed on wet towel paper in
glass Petri dishes under lab conditions. As control, a similar
number of seeds from open pollinated fruits were germinated.
To avoid fungal contamination, we applied a commercial fun-
gicide (Vitabax 40 WP) at the beginning of the experiment.
The seeds were monitored and wetted (if necessary) twice a
week and the number of germinated seeds recorded for two
months. We considered a seed germinated when the radicle
emergence from the seed coat was noticeable.
We performed an ANOVA test to detect signicant differ-
ences in mean seed production between treatments per species
and, after a signicant result, we conducted post hoc pairwise
comparisons (Tukey’s HSD test). Differences in mean cumula-
tive percent of germinated seeds among treatments (self- and
cross-pollinated, and natural pollination) for each species
were evaluated using a non-parametric Kruskall–Wallis test
(Zar, 2010). We used the Wilcoxon test for paired compari-
sons between treatments when signicant differences were
detected and applied a Bonferroni’s correction (Zar, 2010).
Analyses were carried out using the built-in statistical func-
tions available in the R software platform (R Core Team,
2023).
The reduction in tness of selfed progeny was estimated
with the Inbreeding depression index (Charlesworth and
Charlesworth, 1987): IDI = 1 – (Ws/ Wo), where Ws = mean
number of seeds per fruit or percentage of germinated seeds
from manual selng and Wo = mean number of seeds per fruit
or proportion of germinated seeds from manual outcrossing.
An IDI-value = 0 indicates the absence of inbreeding de-
pression, while an IDI value = 1 indicates strong inbreeding
depression.
Reproductive assurance
To estimate the contribution of selng to reproductive suc-
cess, we compared the fruit set between emasculated and
intact owers under open pollination conditions in two con-
secutive owering seasons. We emasculated 474 owers from
13 to 42 plants per species in 2020 and 975 owers from 31
to 53 plants per species in 2021. As control group, a similar
number of intact owers were selected in the same plants.
Since plants from the studied species usually do not reproduce
in consecutive years, the groups of manipulated plants dif-
fered in both years. Using an aluminium ladder, we included
plants on host-trees within reach of six meters in height.
Emasculation was conducted in the afternoon (14–17 h)
before oral anthesis, swollen ower buds in pre-anthesis
were carefully open with a pair of tweezers and the anthers
removed. This manipulation did not alter the oral anthesis.
In the case of species with a high oral display per night, usu-
ally > 1 ower (W. nephrolepis and W. pedicellata), all owers
in anthesis were emasculated to avoid the possibility of gei-
tonogamy. Fruits from emasculated owers indicates a suc-
cessful pollinator visit, whereas fruits from intact owers may
include both autonomous self- and cross-pollination.
We estimated the probability of fruit set between treat-
ments (emasculated vs. control) with a generalized linear
model (GLM) using a binomial distribution (link = ‘logit’)
and a dichotomous response variable (success vs. failure).
The model included as predictor variables: ‘treatment’, ‘year’,
and their interaction, with categories ‘emasculated’ and ‘year
2020’ as reference. The model was estimated with the base
package of the platform R (R Core Team, 2023). The Hosmer
and Lemeshow test (ResourceSelection package; Lele et al.,
2019) evaluated the t of the logistic model to the data. For
those signicant variables, we estimated the ‘odds ratio’ be-
tween the reference and respective categories of each variable
and its 95% condence limits.
The contribution of selng to the reproductive success (i.e.
fruit set) of each species per year was calculated using the
Reproductive Assurance Index (Schoen and Lloyd, 1992):
RAI = (Pi ‐ Pe)/ Pi; where Pi is the proportion of fruits from
intact owers and Pe is the proportion of fruits from emascu-
lated owers. Selng contributes to reproduction if the RAI-
value is greater than zero; when multiplied by 100, it indicates
its relative contribution to the total fruit set. We also estimated
the RAI using data on seed set from the 2021 season. For this,
we counted the number of seeds in a sample of 16–32 fruits
per treatment from each of the four studied species.
Results
Floral biology
Mean ower production per inorescence varied from eight
owers in W. subsecunda to 55 owers in W. pedicellata.
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5Núñez-Hidalgo and Cascante-Marín – Selfing in epiphytic bromeliads compensates for the limited pollination
Depending on the species, one to several owers open per
night, with W. ampla and W. subsecunda being less suscep-
tible to geitonogamy, both species mostly open one ower per
night (Table 1). All species released oral volatiles reminiscent
of fermented fruits or garlic scents, whereas nectar produc-
tion varied in terms of volume (11.9–598.1 μL) and concen-
tration (8–18°Brix) per ower (Table 1).
In all studied species, reproductive organs were exposed
to pollinators, the stigma and anthers projecting from or
close to the corolla mouth (Fig. 1). Herkogamy was ab-
sent in W. nephrolepis and W. subsecunda, but variable in
W. ampla and W. pedicellata, with some plants developing
owers with approach herkogamy (i.e. the stigma longer
than the anthers) (Fig. 1C and 1K). Flowers of W. ampla
and W. nephrolepis were distinguished by the upper portion
of the style curving downward and away from the anthers
(Fig. 1C). The four species showed incomplete protogyny.
The stigma receptivity occurred early, sometimes even at the
bud stage preceding anthesis, but soon it overlapped with
pollen presentation. Temporal separation between female
and male function varied within and between species by up
to 2 h (Table 1).
Flowers exhibited late-afternoon anthesis (15:00–
18:00 h), remaining fully open at night and for a period
from 8 h in W. nephrolepis up to 24 h in W. ampla (Table
1). Flower senescence followed a similar pattern among the
studied species, at the end of the owerʼs life, the corolla
loses its turgor and collapses (Fig. 1 and see Supporting
Information—Videos S1 to S3). In the absence of herkogamy,
the constriction of the petals brings the anthers with re-
maining pollen grains into contact with the stigma, which
is still receptive and has accumulated a viscous uid in the
cupular stigmatic lobes. In W. ampla and W. nephrolepis,
nectar dripping on the lower petal may remove pollen and
deposit it on the stigma, increasing the likelihood of au-
tonomous self-pollination.
Pollinators and floral visitors
The video recording data comprised 454 nights and 1448
monitored owers (see Supporting Information—Table S1).
Bats visited the studied Werauhia on 33 occasions, usually
between 19:00–23:00 h and 01:30–03:30 h, and each visit to
a ower lasted around 2 s. The video images did not allow
a precise identication of the bat species, but they revealed
contact between the batʼs head and the owerʼs reproductive
organs (Fig. 1 and see Supporting Information—Videos S4–
S7). Overall, the visitation rate per night per plant was quite
low (0.07 visits) and varied among years and species from
0 to 0.24 (see Supporting Information—Table S1). In a few
events, the video cameras were activated at night, but no ac-
tivity was documented, which suggests the possibility of un-
recorded visits.
Sporadic visits by the hummingbird Lampornis calolaemus
(Trochilidae) to owers of W. ampla, W. nephrolepis, and W.
pedicellata were also video-recorded during the late after-
noon at the beginning of ower anthesis (16:50–17:20 h)
and the following morning (6:00–8:00 h) when owers
Table 1. Floral biology traits of four sympatric Werauhia species (Bromeliaceae: Tillandsioideae) from a montane forest, Cerros La Carpintera, Costa
Rica.
Floral trait W. ampla W. nephrolepis W. pedicellata W. subsecunda
Flowers per inorescence—mean ± SD
(range, sample size)
13.3 ± 3.6
(7–24, 45)
26.7 ± 6.1
(9–42, 42)
54.7 ± 29.6
(20–145, 42)
8 ± 1.8
(3–13, 50)
Floral display (open owers per da)—
mean ± SD (range, sample size)
1 (rarely 2)
(37)
6.5 ± 2.5
(2–14, 37)
5.4 ± 2.7
(2–12, 32)
1 (rarely 2 or 3)
(62)
Colour of peduncle, primary, and
oral bracts at anthesis
Green to brown Greenish Green with reddish
stripes
Green
Anthesis time Late afternoon
15–17:30 h
(n = 72)
Late afternoon
16–17:30 h
(n = 71)
Late afternoon
16‒18:00 h
(n = 91)
Late afternoon
15:30–17:00 h
(n = 56)
Flower longevity 24 h
(n = 58)
6–8 h
(n = 55)
16–19 h
(n = 31)
15–17 h
(n = 32)
Corolla shape Campanulate Bilabiate Campanulate Campanulate
Corolla colour White-green and suffused with
purple toward the petals
apex
White-greenish White-translucent White-greenish
Herkogamy type,
(anters-stigma separation, sampled
owers)
Absent or approach type,
stigma curved
(2–5 mm, n = 58)
Absent, stigma curved
(n = 55)
Absent or approach
type
(1.5‒2 mm, n = 31)
Mostly absent
(n = 32)
Dichogamy type
(temporal separation, sampled owers)
Protogyny, incomplete
(5‒135 min, 58)
Protogyny, incomplete
(10‒60 min, 55)
Protogyny,
incomplete
(20‒75 min, 31)
Protogyny,
incomplete
(5‒70 min, 32)
Emission of oral scents
(organoleptic test)
Slightly perceptible,
fermented fruits
Perceptible,
garlic and fermented fruits
Perceptible,
garlic
Perceptible,
fermented fruits
Nectar volume (μl) per ower—
mean ± SD, [range], (sample size)
598.1 ± 217.2
[184.2–952.9]
(33 /4 ind)
327.7 ± 199
[30–574.8]
(30 /6 ind)
11.9 ± 10.0
[1–50.5]
(33 /11 ind)
35.3 ± 29
[5–82.5]
(20 /5 ind)
Nectar concentration (ºBrix)—mode,
range (sample size)
17, 12–18
(33 ./4 ind.)
12, 8–14
(30 ./6 ind.)
12, 3–13
(33 ./11 ind.)
12, 8–13
(20 ./5 ind.)
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6AoB PLANTS, 2024, Vol. 16, No. 2
were wilting. A nocturnal and arboreal mouse from genus
Reithrodontomys (Rodentia: Cricetidae) was occasionally
recorded visiting owers of W. ampla and W. nephrolepis.
Stingless bees (Trigona sp., Apidae) were seen on owers of
W. ampla and W. nephrolepis collecting pollen from the an-
thers in the following day of anthesis.
During the mist-netting sampling of eight nights and with
an effort of 675 m2/h, we captured 46 bats from nine genera.
Pollen from the studied Werauhia species was recovered
from three (out of ve) captured individuals of the nectar-
ivorous leaf-nosed bats Hylonycteris underwoodi and from
the single capture of Glossophaga soricina (see Supporting
Information—Table S2). Pollen counts varied between 7
and 5250 grains per sampled individual. Additional pollen
recovered from the bats mainly belonged to the shrubby
epiphytic nightshades: Merinthopodium neuranthum and
Schultesianthus leucanthus (Solanaceae) (see Supporting
Information—Table S2).
Breeding systems
Hand self- and cross-pollinations resulted in high percentages
(>75%) of fully developed fruits, except in W. pedicellata
(50% and 58.1%, respectively) (Table 2). Fruit set from au-
tonomous selng was higher for W. subsecunda (76.7%) and
W. nephrolepis (71.1%) and moderate in W. ampla (43.3%)
and W. pedicellata (31.1%). The breeding systems of the four
Werauhia species are characterized by high values of self-
compatibility (SCI = 0.86–1.14), with relatively high values of
self-fertility (AFI = 0.53–1.00), which indicate a high ability
to self-pollinate by autonomous means. The agamospermy
index suggested a very low degree of potential apomixis in W.
nephrolepis and W. pedicellata (AGI = 0.11 and 0.06, respect-
ively) (Table 2).
Reproductive success and inbreeding depression
The average seed set per fruit did not signicantly differ be-
tween manually self- and cross-pollinated fruits for each spe-
cies (Fig. 2), supporting the high self-compatibility condition
previously recorded using fruit-set data. Moreover, inbreeding
depression effects were absent or low for seed production,
with IDI values ranging from −0.10 to 0.15. Comparing the
number of seeds produced by autonomous selng versus con-
trolled self-pollination revealed no statistically signicant dif-
ferences, indicating the high efcacy of selng at the level of
seed production. (Fig. 2). Similar amounts of seeds were de-
veloped in fruits from open and controlled cross-pollination
(Fig. 2).
Seed germination was high (>80%) and did not differ
statistically between self-, cross-, and open pollinated seeds,
except for W. pedicellata, which selfed seeds had a lower ger-
mination rate (Fig. 3). The studied species experienced null to
low negative effects of inbreeding on their germination cap-
acity, except for W. pedicellata (IDI value = 0.34). In all spe-
cies, seedlings remained alive by the end of the experiment
after two months of sowing.
Reproductive assurance
In all species and in both studied years, emasculated owers
developed fewer fruits compared to intact owers (Figure
4). The GLM results indicated a signicant effect of ‘treat-
ment’, but neither ‘year’ nor their interaction did, except
for W. pedicellata whose response was not consistent across
years (Table 3, Figure 4). The odds ratios indicated that intact
owers capable of autonomous selng had 3.4 times (in W.
ampla) to nearly 12 times (in W. subsecunda) more chances
of producing fruits than emasculated owers that require pol-
linator visits (Table 3).
The estimation of reproductive assurance indicated a con-
tribution of autonomous selng to fruit set from moderate to
high (54–80%), except for W. pedicellata in 2020, whose fruit
set was affected by herbivory (Figure 4). For seed production
in 2021, the contribution of selng to the number of seeds
per capsule was low (RAI ≤ 0.14), except for W. subsecunda
(RAI = 0.48) (Fig. 3).
Discussion
The Bromeliaceae family exhibits a tendency towards
selng, but evidence of its potential adaptive value is
lacking. In this study, we combined data from oral biology,
pollination ecology, and breeding systems to demonstrate
that selng contributes signicantly to the reproductive
success of bromeliads. The studied Werauhia species from
the Tillandsioideae subfamily showed a specialized pollin-
ation system that promotes out-crossing but experienced
low visitation by nectar-feeding bats. The reduced events of
cross-pollination were compensated by autonomous selng
that occurs at the end of the ower’s life and secures the
plant´s reproductive success.
Table 2. Results of controlled pollination treatments and values of indexes that describe the breeding systems of four epiphytic Werauhia species
(Bromeliaceae: Tillandsioideae) in a montane forest, Cerros La Carpintera, Costa Rica. Data are fruit percentages (%) and in parenthesis the number of
developed fruits/manipulated flowers.
Experimenal variable W. ampla W. nephrolepis W. pedicellata W. subsecunda
Number of plants (N) 17 15 16 25
Manual self-pollination 75.0% (12/16) 100% (36/36) 50% (15/30) 87.5% (14/16)
Manual cross-pollination 82.4 % (14/17) 94.3 % (33/35) 58.1 % (18/31) 76.5 % (13/17)
Autonomous self-pollination 43.3% (13/30) 71.1% (32/45) 31.1% (14/45) 76.7% (23/30)
Agamospermy 0 (0/19) 10.5% (4/38) 3.4% (1/29) 0 (0/17)
Self-compatibility index (SCI) 0.91 1.06 0.86 1.14
Auto-fertility index (AFI) 0.53 0.75 0.54 1.00
Agamospermy index (AGI) 0.00 0.11 0.06 0.00
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7Núñez-Hidalgo and Cascante-Marín – Selfing in epiphytic bromeliads compensates for the limited pollination
Specialized pollination and mechanism of selfing
The studied Werauhia species conform to the traditional
bat-pollination syndrome with nocturnal anthesis behaviour
of owers with dull coloration, emission of oral scents as
chemical attractants and diluted nectar in high volume as re-
ward (sensu Faegri and van der Pijl, 1979). The nocturnal
video recording of bats visiting the owers and the captured
bats carrying pollen grains from the studied species conrmed
this specialized pollination system.
Despite demonstrating unambiguous oral adaptations
for cross-pollination, the breeding systems of the studied
Werauhia were highly self-compatible and able to self-fertilize
autonomously. The combination of incomplete protogyny
and a lack of or variable herkogamy (in W. ampla and W.
pedicellata) is likely what facilitates autonomous deposition
of self-pollen on the stigma of the studied species. However,
selng did not appear to occur either before (prior) or during
anthesis (competing) (sensu Lloyd and Schoen, 1992). When
owers were fully open, direct contact between stigma and
anthers was prevented by the stigma’s distinctive cup-shaped
lobes, which served to conceal the receptive area within
(Brown and Gilmartin, 1989; Barfuss et al., 2016). Also, the
ventral torsion of the style near the stigma in W. ampla and
W. nephrolepis may also reduce the chances of stigma-anther
Figure 2. Seed set in four epiphytic Werauhia species (Bromeliaceae: Tillandsioideae) under different pollination treatments. Plants from a montane
tropical forest, Cerros La Carpintera, Costa Rica. Bars are mean number of seeds per fruit and vertical lines are 1 SE. Different letters indicate significant
differences between treatments per species after a Tukey test.
Figure 3. Seed germination capacity of progeny sired from hand self- and cross-pollination and open pollination in four species of Werauhia
(Bromeliaceae: Tillandsioideae) from a montane forest, Cerros La Carpintera, Costa Rica. Data are mean germination percentages from 12 replicates
of 40 seeds per treatment after two-months of monitoring. Vertical lines = 1 SE. Different letters indicate significant differences between treatments
per species after a Wilcoxon’s test. The estimated values of the inbreeding depression index were for W. ampla = -0.13, W. nephrolepis = 0.00, W.
pedicellata = 0.34 and W. subsecunda = 0.02.
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8AoB PLANTS, 2024, Vol. 16, No. 2
contact during anthesis. This feature of the style and stigma is
found in other species of Werauhia, and the degree of torsion
varies (Utley, 1983), but its potential signicance to pollin-
ation has not been discussed previously.
Rather, we found that autonomous selng in the studied
Werauhias occurred at the end of the ower’s life. The pat-
tern of ower senescence by which the corolla closes and
forces the anthers with exposed pollen into contact with the
still-receptive stigma corresponds to the mechanism of ‘cor-
olla closure’ described by Goodwillie and Weber (2018).
The stigmatic exudate that visibly accumulates in the stigma
lobes of the studied species probably helps the pollen grains
stick when the corolla closes. Following this evidence, self-
pollination in the studied Werauhia would represent a mech-
anism of ‘delayed selng’ (sensu Lloyd and Schoen, 1992),
and it suggests that reproductive assurance rather than re-
productive isolation is its primary benet, as the latter would
most likely select for earlier or preemptive selng to prevent
bats from depositing heterospecic pollen onto the stigmas
(sensu Randle et al., 2016).
Within the Bromeliaceae family, chiroterophily is present in
subfamily Pitcairnioideae (Pitcairnia) but is better represented
in Tillandsioideae, mainly in Pseudoalcantarea and Vriesea,
and Werauhia is thought to be the genus with the greatest spe-
cialization in bat pollination (reviewed by Aguilar-Rodríguez
et al., 2019a). Fenster and Martén-Rodríguez (2007) suggested
that specialized pollination is frequently associated with oral
mechanisms to self-pollinate; however, several examples in-
dicate that for bat pollination such association is weak. In
a group of bat-pollinated gesneriads, Martén-Rodríguez and
Fenster (2010) found they were unable to self-pollinate au-
tonomously, while hummingbird-pollinated species exhibit
high potential for autonomous selng. Additional examples
of neotropical chiropterophilous plants evidence the pres-
ence of self-incompatibility mechanisms or the inability to
self-pollinate autonomously (e.g. Sazima and Sazima, 1978;
Gibbs et al., 1999; Gribel and Gibbs, 2002; Sazima et al.,
2003) suggesting that specialization in pollination and oral
traits that promote selng are not necessarily associated in an
evolutionary context (Fenster and Martén-Rodríguez 2007).
Thus, the high frequency of Werauhia species and brome-
liads, in general, with specialized pollination systems and high
selng ability might be a particularity of this plant lineage.
Pollinators and pollinator limitation
Two nectar-feeding bat species from the subfamily
Glossophaginae (Glossophaga soricina and Hylonycteris
underwoodi) represent the most probable pollinators of
the studied species. Our census was limited in scope (two
months), but according to a more extensive survey of the
bat community (Durán, 2013), a third nectarivorous species
(G. commissarisi) is present in our study site. Based on the
frequency of captures, our data suggest that Hylonycteris
underwoodi is likely the most important pollinator of the
studied epiphytic bromeliads. This is a small nectarivorous
bat distributed from Mexico to Panama in primary and older
secondary forests and from sea level to 2640 m asl (Wilson
and Mittermeier, 2019).
Figure 4. Fruit set from emasculated (E) and intact (C) flowers under open pollination conditions of four Werauhia (Bromeliaceae: Tillandsioideae)
species in a montane forest, Cerros La Carpintera, Costa Rica. Bars represent the proportions of developed fruits per treatment in two consecutive
reproductive seasons (2020 and 2021). The value of the Reproductive Assurance Index (RAI) is indicated for each species and year. The sample size
(number of flowers) per treatment is indicated at the bottom of each column.
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9Núñez-Hidalgo and Cascante-Marín – Selfing in epiphytic bromeliads compensates for the limited pollination
Plants with specialized pollination systems are prone to
pollen limitation due to unpredictable visitation by their pol-
linators (Knight et al., 2005; Martén-Rodríguez and Fenster,
2010). In chiropterophilous plants, bats are considered ‘good’
pollinators because they carry large amounts of pollen from
different paternal genotypes and can disperse it over long dis-
tances (Fleming et al., 2009). In spite of this, our evidence
from camera traps suggests a low pollinator availability of
nectar-feeding bats, with visitation ranging from none to 0.24
visits per night per plant. Data from other bat-pollinated bro-
meliads suggest varying but usually higher visitation rates;
for instance, Aguilar-Rodríguez et al. (2019b) found no visit-
ation to Werauhia nutans but up to 4.2 visits per ower per
night in Pseudalcantarea viridiora. While in W. gladioliora,
Tschapka and von Helversen (2007) observed 1–44 visits per
ower per night. Bat visits to owers can be quite fast (less
than 0.5 s) and it is possible that camera traps have under-
estimated the visitation rate. However, our ower emascula-
tion experiment, which resulted in low reproductive success
(<26% fruit set), supports the idea of limited pollinator
services in the studied epiphytic bromeliads.
The low pollinator visitation recorded may arise from the
interaction of several ecological factors acting locally. A low
diversity of pollinators has been associated with increased
pollen limitation (Knight et al., 2005). Species richness in
nectar-feeding bat communities shows a decreasing pattern
with respect to elevation (Fleming et al., 2005), with fewer
species in montane forests compared to lowland habitats. The
absence of Anoura geoffroyi (Phyllostomidae) at the study
site is notable since it is a nectarivorous species from montane
forests and considered abundant throughout its distribution
range (Ortega and Alarcón-D., 2008). In Costa Rica, however,
it is an uncommon and rarely captured species, although it is
apparently common in some localities (LaVal and Herrera-R.,
2002; Wainwright, 2007). The lower diversity (three species)
of pollinating bats in our research site, located at around
1700 m asl, compared to a Costa Rican lowland bat com-
munity with four nectarivorous species (Tschapka and von
Helversen 2007), presumably plays a role in the limited visit-
ation we recorded.
Low oral visitation may also be indicative of a low popu-
lation density of pollinators. Hylonycteris underwoodi is
a rare species that never occurs in dense populations and
roosts in small groups of one to four individuals (Wilson
and Mittermeier, 2019). In a lowland bat community, this
bat species was unfrequently captured in mist nests and rep-
resented 4% of the captures (Tschapka and von Helverson,
2007). Similarly, in a previous bat inventory at our study site
and with a sampling effort spanning a whole year (39 nights
and 21 060 m2/h), Durán (2013) documented only ve H.
underwoodi individuals from a total of 142 captured bats
(3.5% of the captures). Overall, the evidence strongly sug-
gests that H. underwoodi has a low population density at our
montane research site, which likely accounts for the observed
low visitation frequency to bromeliad owers. According to
Fleming et al. (2005), nectarivorous bats density is probably
Table 3. Parameter estimates for the generalized lineal models on the production of fruits between emasculated and unmanipuled flowers under open-
pollination conditions in four Werauhia species (Bromeliaceae: Tillandsioideae) in a montane forest, Cerros La Carpintera, Costa Rica. The reference
categories are “emasculated” and “2020” for Treatment and Year, respectively.
Parameters by species d.f. Estimate S.E. Wald chi-square P-value Odds ratio Condence interval (95%)
W. ampla
Intercept 1 −1.16 0.26 −4.54 <0.001
Treatment 1 1.22 0.33 3.76 <0.001 3.40 1.80–6.44
Year 1 0.10 0.30 0.34 0.732 1.11 –
Treatment × year 1 0.03 0.39 0.09 0.929 1.03 –
Error 583
W. nephrolepis
Intercept 1 −1.29 0.20 −6.38 <0.001
Treatment 1 2.32 0.28 8.37 <0.001 10.17 5.9–17.51
Year 1 −0.17 0.25 −0.68 0.498 0.84 –
Treatment × year 1 0.48 0.35 1.38 0.164 1.62 –
Error 880
W. pedicellata
Intercept 1 −1.09 0.21 −5.24 <0.001
Treatment 1 0.28 0.29 0.99 0.321 1.33 –
Year 1 −0.44 0.26 −1.70 0.088 0.65 –
Treatment × year 1 1.12 0.34 3.28 0.001 3.07 1.57–6.01
Error 846
W. subsecunda
Intercept 1 −1.97 0.28 −7.16 <0.001
Treatment 1 2.48 0.32 7.68 <0.001 11.96 6.34–22.53
Year 1 0.37 0.34 1.09 0.278 1.45 –
Treatment × year 1 −0.09 0.42 −0.21 0.831 0.92 –
Error 614
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10 AoB PLANTS, 2024, Vol. 16, No. 2
low in most habitats; however, the aforementioned research
by Tschapka and von Helversen (2007) also revealed a higher
abundance of bats that frequently visited the owers of W.
gladioliora in a lowland forest. This spatial variation in pol-
linator abundance may affect the efciency of selng as a re-
productive assurance mechanism.
Factors related to habitat fragmentation may, in turn, af-
fect the density of resident bat pollinators (Steffan-Dewenter
and Tscharntke, 1999; Cunningham, 2000; Liu and Koptur,
2003; Knight et al., 2005) and negatively impact pollination
services. The studied montane forest is a medium-sized forest
fragment (ca. 2.400 ha) loosely connected to major forested
areas in the much larger Talamanca Mountain range. This
condition may limit long-distance migration, affect the sta-
bility of the local population of H. underwoodi, or impede
the establishment of other nectarivorous species such as
A. geoffroyi. In addition, pollen grains of non-bromeliad
plants recovered from bats suggest that inter-specic com-
petition among co-owering bat-pollinated plants may be
a potential cause of decreased visitation. On the contrary,
intra-specic competition for pollinators among sympatric
Werauhia is likely low, since the investigated species exhibit
a staggered owering phenology in the study site (Cascante-
Marín et al., 2017). Furthermore, this phenological pattern
may be an indicator that reproductive isolation is not the
primary function of selng but rather its reproductive as-
surance function.
Reproductive assurance
The mechanism of autonomous delayed selng of the studied
Werauhia was key to their reproductive success, representing
54–80% of the total fruit set. Recording the time of selng
in bromeliad pollination studies is not a common practice
(Cascante-Marín and Núñez-Hidalgo 2023), but the few
studies that have reported delayed selng in bat-pollinated
and highly autofertile bromeliads belong to Werauhia spe-
cies (Cascante-Marín et al., 2005; Aguilar-Rodríguez et al.,
2019b). However, these studies did not assess its contribution
to reproductive success.
The establishment and persistence of selng are counter-
acted by the negative effects of inbreeding (Charlesworth
and Charlesworth, 1987). Theoretically, the maintenance of
selng would occur when the adequacy of the selfed progeny
surpasses that of outcrossed origin by a factor of WS/WO > 0.5
(Herlihy and Eckert, 2002; Eckert et al., 2006). We found that
inbreeding depression at early stages of the progeny had low
or null effects on the number of seeds (IDI-values ≤ 0.15)
and germination capacity (IDI-values ≤ 0.34) of selfed seeds.
This likely contributes to the maintenance of selng in the
studied Werauhia populations. However, life-time estimations
of inbreeding depression would conrm or reject the positive
effects of selng and its evolutionary stability (Delmas et al.,
2014).
In oral emasculation experiments, reproductive assurance
may be overestimated due to low visitation caused by modi-
cations to ower attractiveness (Eckert et al., 2006). In our
case, anthers removal may have caused a minor alteration
to the ower’s visual appearance, and we presume a non-
signicant effect since it has been demonstrated that nectar-
feeding bats depend more on olfactory and acoustic cues
when searching for nocturnal owers (Gonzalez-Terrazas et
al., 2016). Also, bats appear to rely more on olfaction when
owers are situated against a complex background (Muchhala
and Serrano, 2015), as is the case with epiphyte plants in the
forest canopy.
Comparable data on manipulative experiments involving
other bromeliads, as well as tropical plants in general, are se-
verely lacking (see Eckert et al., 2006; Busch and Delph 2012).
Lasso and Ackerman (2004) found that emasculated owers
of the hummingbird-pollinated Werauhia sintenisii from the
island of Puerto Rico experienced low pollinator visitation.
The authors suggested the value of selng in the reproduction
of this species. Studies from temperate zone plants are more
prevalent in the literature (e.g. Eckert et al., 2006, Kalisz et
al., 2004; Moeller 2006; Brys and Jacquemyn 2011; Yang et
al., 2018; Teixido and Aizen 2019) and show that the effect of
selng on reproductive success exhibits temporal and spatial
variation. The few studies on tropical plants have found that
the contribution of selng to reproductive assurance may vary
among plants with different pollination systems in a group of
gesneriads (Martén-Rodríguez and Fenster, 2010). It was also
found that the contribution of selng to the reproduction of
the vine Ipomoea hederacea (Convolvulaceae) varied among
reproductive seasons (Delgado-Dávila and Martén-Rodríguez
2021).
Selng capacity and the degree of self-compatibility in
Bromeliaceae are positively associated, with some of the
variation explained by oral biology attributes such as
anthers-stigma separation or herkogamy (Cascante-Marín
and Núñez-Hidalgo, 2023). We found that W. ampla and W.
pedicellata were highly self-compatible (SCI = 0.91 and 0.86,
respectively) but exhibited lower selng capacity (AFI = 0.53
and 0.54, respectively), which resulted in lower contribution
to reproduction assurance. These differences in selng cap-
acity can be explained by the observed variation in herkogamy
in the studied populations that may reduce the effectiveness
of the selng mechanism of corolla closure. Previous studies
have shown that autofertility is correlated with variations in
herkogamy; furthermore, this oral trait exhibits partitioning
primarily between populations (Moeller, 2006). The variation
of this oral trait is poorly documented in tropical plants, and
it has been suggested that it can evolve rapidly in response to
environmental changes affecting cross-pollination (Opedal et
al., 2017; Opedal 2018).
Ecological factors may offset the benecial effects of selng,
as shown by the contrasting outcomes of the reproductive
assurance estimation in W. pedicellata, despite its moderate
selng capacity (AFI = 0.54). This unexpected result can
be explained by herbivory caused by larvae of a buttery
(Lepidoptera: Lycaenidae) that consumed early-developing
capsular fruits in several plants during the 2020 season.
Herbivory of reproductive structures may alter the repro-
ductive success of plants, as documented in other bromeliad
species (Cascante-Marín et al., 2008; Orozco-Ibarrola et al.,
2015). This particular situation likely accounts for the lower
fruit set in open pollination recorded in the rst owering
season studied. This type of herbivory may result in complete
loss of a plant´s inorescence, as observed in plants kept in a
greenhouse and eld conditions.
Concluding remarks
This study provides novel evidence of the function of de-
layed selng as a reproductive assurance mechanism in the
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11Núñez-Hidalgo and Cascante-Marín – Selfing in epiphytic bromeliads compensates for the limited pollination
species-rich family Bromeliaceae, a plant lineage character-
ized by a tendency towards self-fertilization. The alternative
hypothesis of selng as a mechanism of reproductive isolation
is not supported because of the non-overlapping owering
seasons previously reported for the investigated Werauhia
species in the study site (Cascante-Marín et al., 2017), which
precludes heterospecic pollen transfer. Moreover, it has been
demonstrated that delayed selng is an ineffective barrier
against hybrid fertilization (Brys et al., 2016). A comprehen-
sive study of potential isolation mechanisms will conrm this
assumption.
We conclude that reproductive success in the studied Werauhia
species is pollinator-limited due to the low visitation rate of its
main bat pollinator. The delayed-selng mechanism is strength-
ened by the lack of inbreeding depression and substantially con-
tributes to reproductive success, compensating for the limited
cross-pollination services provided by nectar-feeding bats. This
selng mode may be common among chiropterophilous bro-
meliads; however, the documented reproductive benets may
vary depending on the ecological context of pollination. Some
reports of delayed selng in predominantly ornithophilous bro-
meliad genera, such as Tillandsia (Orozco-Ibarrola et al., 2015)
and Pitcairnia (Wendt et al., 2002), warrant further investi-
gation to test whether selng as a mechanism of reproductive
assurance has also evolved in bromeliad lineages with other
specialized pollination systems than chiropterophily.
The high prevalence of selng in Bromeliaceae suggests a
potential ecological and evolutionary advantage. Unveiling
such benets requires detailed studies combining oral
biology, breeding systems, and pollination in bromeliads and
other tropical plants. Manipulative experiments that encom-
pass temporal and spatial variation in pollination conditions
may help us understand the ecological factors that shape the
effects of selng in tropical plants.
Supporting Information
The following additional information is available in the on-
line version of this article –
Appendix. Raw data from experiments.
Table S1. Visitation data of nectarivorous bats to
owers of four epiphytic bromeliads from genus Werauhia
(Bromeliaceae: Tillandsioideae) in a montane forest, Cerros
La Carpintera, Costa Rica. Data from six video-camera traps
from the owering periods of 2019, 2020, and 2021.
Table S2. Number of pollen grains per plant species re-
covered from the six nectarivorous bats captured in a mon-
tane forest, Cerros La Carpintera, Costa Rica. Data from a
sampling effort of 675 m2/h during eigth nights from January
to February 2020.
Video S1. Time-lapse video of a ower senescence of
Werauhia ampla (Bromeliaceae). Frame rate: 30 fps. Duration:
15 s.
Video S2. Time-lapse video of a ower senescence of
Werauhia subsecunda (Bromeliaceae). Frame rate: 30 fps.
Duration: 27 s.
Video S3. Time-lapse video of a ower senescence of
Werauhia nephrolepis (Bromeliaceae). Frame rate: 30 fps.
Duration: 16 s.
Video S4. Slow motion video (10×) of a bat visiting a noc-
turnal ower of Werauhia ampla (Bromeliaceae). Duration:
22 s.
Video S5. Slow motion video (10×) of a bat visiting a
nocturnal ower of Werauhia nephrolepis (Bromeliaceae).
Duration: 7 s.
Video S6. Slow motion video (10×) of a bat visiting a
nocturnal ower of Werauhia pedicellata (Bromeliaceae).
Duration: 10 s.
Video S7. Slow motion video (10×) of a bat visiting a
nocturnal ower of Werauhia subsecunda (Bromeliaceae).
Duration: 10 s.
Acknowledgments
This study was conducted in partial fulllment of the require-
ments of the Master degree of Stephanie Núñez-Hidalgo at the
Graduate Program ‘Sistema de Estudios de Posgrado’ from the
Universidad de Costa Rica. The Vicerrectoría de Investigación
from Universidad de Costa Rica provided nancial support
(Proyect C0-060 to ACM). The authors would like to thank
the staff of Iztarú Field School and the Association of Guides
and Scouts of Costa Rica for granting permission to conduct
this research in their facilities and Jorge Gonzalez who greatly
assisted in capturing and identifying bats. The authors also
acknowledge the valuable comments and suggestions made
by two anonymous reviewers that improved the quality of
the manuscript.
Contributions by the Authors
Both authors designed the conceptual framework of the study.
S.N.H. collected most of the data and led the analysis and in-
terpretation of the data with support of A.C.M., while S.N.H.
wrote the initial drafts of the manuscript. A.C.M. contributed
to the nal version of the manuscript and gave the nal ap-
proval for publication.
Conflict of Interest Statement
The authors declare no conict of interest.
Data Availability
The data underlying this article are available in the article and
in its online Supporting Information.
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