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The consequences of flower colour polymorphism on the reproductive success of a neotropical deceptive orchid

March 2025
Plant Biology

DOI:10.1111/plb.70020

Authors:

Beatriz Lucas Arida

State University of Campinas (UNICAMP)

Fábio Pinheiro

State University of Campinas (UNICAMP)

Lucrezia Laccetti

University of Naples Federico II

Maria Gabriela Gutierrez Camargo

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Deceptive plants often exhibit elevated levels of polymorphism. The basis of the association between flower polymorphism and deceptive strategies, however, remains unclear. Epidendrum fulgens , a Neotropical deceptive orchid pollinated by butterflies, has an unexplored intrapopulation flower colour polymorphism. Here, we investigate the consequences of this polymorphism on its reproductive success. We performed field and common garden experiments, aiming to detect pollinator‐mediated selection strength and direction over time, and test whether the presence of multiple colour morphs increases species' reproductive success. In the field, we monitored plant reproductive success and floral morphology on two populations over two flowering seasons and performed selection gradient analyses. In the common garden, we assembled plots of cultivated plants with same and different flower colour individuals (i.e., mono‐ and polymorphic plots), exposed them to pollinators and monitored their reproductive success. In both sites we also monitored the local pollinator community. In the field, colour morphs performed equally, but we found coherences between morphological differentiation and the direction of selection, which was very dynamic. In the common garden, mono‐ and polymorphic plots also performed equally, with highly variable reproductive success over time. We also found a highly diverse pollinator community. Our results suggest that flower polymorphism in E. fulgens is maintained by a combination of factors, including varying pollinator‐mediated selection, assortative mating due to differential pollinator preferences and different phenotype heritability. Natural selection varied across time and space, indicating a dynamic interplay between pollinators and flower morphs.

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RESEARCH ARTICLE

The consequences of ﬂower colour polymorphism on the

reproductive success of a neotropical deceptive orchid

B. L. Arida

, F. Pinheiro

, L. Laccetti

, M. G. G. Camargo

, A. V. L. Freitas

& G. Scopece

1 Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Brazil

2 Department of Biology, University of Naples Federico II, Complesso Universitario MSA, Naples, Italy

3 Center for Research on Biodiversity Dynamics and Climate Change and Department of Biodiversity, Phenology Lab, S˜

ao Paulo State University, Biosciences

Institute, Rio Claro, Brazil

4 Departamento de Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Brazil

Keywords

Epidendrum; ﬂoral traits; ﬂuctuating selection;

food deception; phenotypic polymorphism;

pollinator-mediated selection.

Correspondence

F. Pinheiro, Departamento de Biologia

Vegetal, Instituto de Biologia, Universidade

Estadual de Campinas, Campinas, SP 13083-

862, Brazil.

E-mail: biopin@unicamp.br

Editor

Z.-.X. Ren

Received: 29 October 2024;

Accepted: 24 February 2025

doi:10.1111/plb.70020

ABSTRACT

•Deceptive plants often exhibit elevated levels of polymorphism. The basis of the associ-

ation between ﬂower polymorphism and deceptive strategies, however, remains

unclear. Epidendrum fulgens, a Neotropical deceptive orchid pollinated by butterﬂies,

has an unexplored intrapopulation ﬂower colour polymorphism. Here, we investigate

the consequences of this polymorphism on its reproductive success.

•We performed ﬁeld and common garden experiments, aiming to detect

pollinator-mediated selection strength and direction over time, and test whether the

presence of multiple colour morphs increases species’ reproductive success. In the ﬁeld,

we monitored plant reproductive success and ﬂoral morphology on two populations

over two ﬂowering seasons and performed selection gradient analyses. In the common

garden, we assembled plots of cultivated plants with same and different ﬂower colour

individuals (i.e., mono- and polymorphic plots), exposed them to pollinators and

monitored their reproductive success. In both sites we also monitored the local pollina-

tor community.

•In the ﬁeld, colour morphs performed equally, but we found coherences between mor-

phological differentiation and the direction of selection, which was very dynamic. In

the common garden, mono- and polymorphic plots also performed equally, with

highly variable reproductive success over time. We also found a highly diverse pollina-

tor community.

•Our results suggest that ﬂower polymorphism in E. fulgens is maintained by a combi-

nation of factors, including varying pollinator-mediated selection, assortative mating

due to differential pollinator preferences and different phenotype heritability. Natural

selection varied across time and space, indicating a dynamic interplay between pollina-

tors and ﬂower morphs.

INTRODUCTION

Intraspeciﬁc variation in phenotypic traits is widespread in

nature. Darwin emphasised the general presence of this intra-

speciﬁc variation, considering it as the raw material upon

which natural selection might act (Darwin 1859). In general,

phenotypic variation is fuelled by genetic recombination and

mutation and is eroded by natural selection that eliminates all

but the most favourable genetic combinations (Stebbins 1950).

Therefore, intraspeciﬁc variation found in natural populations

should result from an equilibrium between the genetic pro-

cesses generating variation and the selecting forces leading to

trait homogeneity (Ellegren & Galtier 2016). An extreme case

of phenotypic variation occurs when individuals of a species

fall into two or more categories, a condition referred to as phe-

notypic polymorphism (Westerband et al.2021).

Flowers, i.e. the sexual organs of Angiosperms, are an ideal

model to investigate the origin and the maintenance of pheno-

typic polymorphisms, as they are particularly variable within

populations (e.g. Darwin’s book The different forms of ﬂowers

on plants of the same species, Darwin 1877). Among different

ﬂoral traits, colour is one of the most polymorphic (Sapir

et al.2021). This trait is typically used by pollinators as a signal

to identify nectar or pollen sources (van der Kooi et al.2021).

In most Angiosperms, ﬂowers within populations have weakly

variable colours (i.e. monomorphic species), because plant

individuals that pollinators are able to recognise as conspeciﬁc

are more easily visited (e.g. ﬂower constancy; Waser 1986). In

these plant–pollinator relationships, plants are continuously

exposed to a choice of pollinators, which imposes directional

selection on ﬂower traits (Schiestl & Johnson 2013), hence

eroding intraspeciﬁc variation. This ﬂower constancy is

expected to be advantageous for both involved parties: as it

increases plant reproductive success by more efﬁcient pollen

transfer between conspeciﬁc individuals, and it increases polli-

nator foraging efﬁciency as searching and learning to handle

new ﬂoral types is costly (J.B. 1963; Chittka et al.1999; Goul-

son 2000). This is the most common mechanism by which

Plant Biology

Plant Biology ISSN 1435-8603

pollinator-mediated natural selection on ﬂoral traits maintains

intraspeciﬁc cohesion and reduces ﬂoral variation (Waser &

Price 1981).

Despite this general situation, however, in many plant spe-

cies ﬂowers exhibit strong discrete or continuous colour varia-

tion (i.e. polymorphic species). Discrete polymorphism in

ﬂower colour is likely to result from genetic variation at a sin-

gle or a few loci (Wu et al.2013), whilst continuous variation

is likely to result from a multigenic architecture with additive

effects or from differential expression of genes involved in pig-

ment biosynthesis (Davies et al.2012; Scopece et al.2020).

Despite an intense research effort, the mechanisms that create

and maintain ﬂower colour polymorphisms within plant spe-

cies are still not completely understood and are highly debated

(Sapir et al.2021). Competing hypotheses are that colour varia-

tion can be maintained due to balancing selection exerted by

multiple selection regimes, ﬂuctuating selection over time and

space, heterozygote advantage and frequency-dependent selec-

tion (Kellenberger et al.2019; Sapir et al.2021 and references

therein). A lack of selection may also maintain variation,

although this currently remains almost untested (but see Jac-

quemyn & Brys 2020).

A peculiar situation is a category of plant species that offer no

reward to their pollinators, known as deceptive plant species

(Sprengel 1793). This pollination strategy evolved in several

plant families but is particularly frequent in Orchidaceae, where

an unusually high proportion of species employ deceptive polli-

nation strategies (Shrestha et al.2020). Unlike rewarding species,

in deceptive species ﬂoral constancy is not expected because,

after a few unrewarded visits, pollinators tend to avoid the

rewardless phenotype (pollinator avoidance learning; Hein-

rich 1975; Dukas & Real 1993; Smithson & Macnair 1997;Raine

&Chittka2007). Thus, in these pollination strategies,

pollinator-mediated natural selection is not expected to elimi-

nate ﬂoral variation, potentially leading to high intraspeciﬁc

ﬂower polymorphisms. Accordingly, higher rates of intraspeciﬁc

phenotypic variability in deceptive orchid species compared to

rewarding ones have been reported (Heinrich 1975;Salzmann

et al.2007;Ackermanet al.2011). The traditional hypothesis to

explain this elevated phenotypic polymorphism in deceptive spe-

cies is that it would slow down pollinator avoidance learning

ability thus increasing plant reproductive success (Heinrich 1975;

Nilsson 1992; Smithson & Macnair 1997;Ferdyet al.1998). This

hypothesis was recently tested in a manipulative experiment by

Aguiar et al.(2020), who corroborated it with a bee pollinated

species, showing how the colour variation of a deceptive orchid

inﬂuences the pollinator’s cognitive aspect of deception. How-

ever, in a meta-analysis of studies conducted to test this hypothe-

sis, Juillet & Scopece (2010) found no relationship between

ﬂower polymorphism and increased reproductive success. Alter-

native explanations raised to justify this elevated polymorphism

in deceptive orchids emphasise a negative frequency-dependent

selection (Gigord et al.2001), a weak selection on ﬂoral traits

(Jacquemyn & Brys 2020), or a directional but ﬂuctuating selec-

tion on ﬂoral traits in different ﬂowering seasons (Scopece

et al.2017). The origin of ﬂower colour polymorphism in decep-

tive species has also been associated with inaccurate colour dis-

crimination by pollinators (Kagawa & Takimoto 2016). In fact,

studies have indicated varied selection patterns as responsible for

the maintenance of ﬂoral polymorphisms, such as the evident

pattern found by Gigord et al.(2001)inDactylorhiza sambucina

(L.) So`o, an example of a deceptive orchid with colour morphs

maintained by pollinator-mediated selection.

The lack of concordance in studies attempting to explain the

reasons that form the basis of the elevated phenotypic poly-

morphism in deceptive orchids calls for additional case studies,

particularly in under-investigated regions and groups. Here,

using the colour polymorphic Neotropical orchid Epidendrum

fulgens Brongn., we investigated the intensity and direction of

pollinator-mediated natural selection over two consecutive

ﬂowering seasons in natural populations, and the relationship

between ﬂower colour polymorphism and reproductive success

components in common garden conditions and in natural

populations. Using these data, we speciﬁcally asked the follow-

ing questions:

1Is ﬂower colour variation discrete in E. fulgens?

2If yes, do different ﬂower colour morphs experience differ-

ent reproductive success during their ﬂowering season in

natural populations?

3Are different colour morphs exposed to different

pollinator-mediated selection?

4If yes, does this reﬂect among-morph morphological

differentiation?

5Does the presence of different colour morphs affect repro-

ductive success?

MATERIAL AND METHODS

Study species, experimental design and characterisation of

colour polymorphism

Epidendrum fulgens is a Neotropical orchid species in the sub-

tribe Laeliinae. This species is found on open sand dune vegeta-

tion, growing directly on sandy soils along the south and

southeastern Brazilian coast (de Mattos et al.2023). It has a

long ﬂowering season during the time of the year with the

highest rainfall, with peak ﬂowering between October and

April. This species does not have ﬂoral nectaries and there are

no records of ﬂoral fragrance, so that there is no correlation

between ﬂoral colour and fragrance, as observed in other

deceptive orchids, such as by Aguiar et al.(2021). It employs a

generalised food-deceptive strategy to attract pollinators, which

are exclusively butterﬂies (Moreira et al.2008; Fuhro

et al.2010; Pinheiro et al.2010) that are primarily visually ori-

ented and able to discriminate yellow-to-red colours (Swihart

& Swihart 1970; Arikawa 2003; Blackiston et al.2011). The pol-

linators of E. fulgens are also essential for pollen transfer (Pin-

heiro et al.2015) and fruit development, with no evidence of

spontaneous autogamy (Fuhro et al.2010). E. fulgens has evi-

dent intrapopulation variation in ﬂower colour, with petals

and sepals ranging from yellow to red, with orange intermedi-

ates (Fig. 1).

With the aims of understanding the mechanisms that main-

tain intrapopulation polymorphism and their consequences on

reproductive success (questions ii,iii and iv), we performed

ﬁeld observations in natural populations of E. fulgens (hereaf-

ter, natural populations survey). Then, in order to understand

whether the presence of different ﬂower colour morphs has an

effect on reproductive success (question v), we set up an exper-

iment manipulating the presence of different colour morphs in

a common garden setup (hereafter, plot experiment). In both

Plant Biology

Colour polymorphism in Epidendrum Arida, Pinheiro, Laccetti, Camargo, Freitas & Scopece

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activities, reproductive success was then estimated. Following

Scopece et al.(2015), pollinia removal was used as a proxy for

male reproductive success (hereafter MRS) and proportion of

fruit formation was used as a proxy for female reproductive

success (hereafter FRS). Here, we focus on the visual aspect of

E. fulgens and choose not to add an analysis regarding the

importance of its ﬂoral scent in attracting pollinators.

Although there is evidence that ﬂoral scent serves as a signal

for attraction of butterﬂies in some cases, as pointed out by

Andersson et al.(2002) and Chen et al.(2021), there is no evi-

dence of ﬂoral scent in E. fulgens and the main known pollina-

tors of the species are mainly visually oriented.

To characterise ﬂower colour polymorphism, we collected

reﬂectance data of the labellum, petal, lateral sepal and dorsal

sepal of 21 ﬂowers from different individuals, seven of each

extreme colour (yellow and red) and seven intermediates

(orange), with an Ocean Optics USB4000 Fibre Optic Spectrom-

eter (Jaz Modular Optical Sensing Suite), allowing detection of

visible and ultraviolet wavelengths and covering the sensitivity

of the photoreceptors of different species of butterﬂy (van der

Kooi et al.2021). Using these data, we validated the three colour

morph categories of E. fulgens: yellow, orange and red (hereafter,

Y, O and R) (e.g. Dalrymple et al.2015) and its polymorphism

as discrete. The results supporting this conclusion are detailed in

the Results, subsection Flower phenotype polymorphism.

Natural populations survey

Field activities were carried out in two natural populations

with similar climate (de Mattos et al.2023) and soil (de Lima

et al.2024) conditions (Bertioga and Ilha do Cardoso, both in

the coastal region of S˜

ao Paulo State, Brazil, hereafter BE and

CA, respectively). To estimate frequency of the three ﬂower

colour morphs, we used the transect method proposed by Cot-

tam & Curtis (1956) and took note of the number of individ-

uals of each morph in an area of 400 m

. This estimation was

carried six times along the ﬂowering phenology of the

investigated species (four times in BE and twice in CA). In both

populations, we labelled 15 individuals from each colour

morph. Between December 2021 and April 2023, a total of 14

ﬁeld expeditions in the natural populations were carried out

(ten to BE and four to CA). On the different expeditions, we

followed the same labelled individuals. Over time, however, if

some of the labelled individuals were lost, we added new indi-

viduals to retain the original sample size. By doing so, a total of

70 individuals were monitored in BE and 68 in CA. For each

labelled individual, we recorded eight morphological traits

potentially involved in pollinator attraction: petal length

(PetL), petal width (PetW), labellum area (LabA), labellum

shape (LabS), lateral sepal length (LatSepL), lateral sepal width

(LatSepW), dorsal sepal length (DorSepL), dorsal sepal

width (DorSepW). For these measurements, we collected one

ﬂower from each labelled individual in each ﬂowering season,

dissected ﬂoral part and distended them between transparent

sheets to avoid 3D deformation. We then took a digital scan of

each ﬂower using a tabletop scanner (HP Scanjet 4670), at a

resolution of 1200 ×1200 dpi with a reference scale on the

back. Calibrated images were thus used to extract ﬂower trait

measurements using ImageJ 1.33 (https://imagej.nih.gov/ij/).

LabS was obtained instead by performing a landmark-based

geometric morphometric analysis. For this, we selected a set of

23 landmarks and semi-landmarks (e.g. Bookstein 1997;

Tyteca 2000) using TpsUtil and TpsDig 2 (Rohlf 2015). The

coordinates of the landmarks and semi-landmarks of the label-

lum were superimposed using a Generalised Procrustes Analy-

sis (GPA) (Rohlf & Slice 1990). Then, we performed a

Principal Components Analysis (PCA) and PC values were

mapped back onto labellum morphology using eigenvectors

(i.e. a set of vectors associated with the set of landmarks and

semi-landmarks and their variation in direction and strength).

Finally, on each ﬁeld expedition, data on MRS and FRS were

recorded on each labelled individual.

To obtain an overview of Lepidoptera characterising the E.

fulgens ﬂower visitors and to assess possible changes in its

Fig. 1. Inﬂorescences of Epidendrum fulgens of the three colour morphs: yellow, orange and red (A). Reﬂectance average (line) and standard deviation (line

shade) spectra of each measured ﬂoral part (from left to right: labellum, petal, lateral sepal and dorsal sepal) of the three morphs (represented by their respec-

tive colour) (B).

Plant Biology

Arida, Pinheiro, Laccetti, Camargo, Freitas & Scopece Colour polymorphism in Epidendrum

composition and abundance over time, we performed direct

observations (for a total of 80 h) using the approach described

in Fuhro et al.(2010). Observations were conducted during

daytime (from 06:00 to 16:00 h) since E. fulgens is pollinated

only in daylight (Fuhro et al.2010). All butterﬂies found were

recorded and classiﬁed into one of the three following catego-

ries: (a) pollinators (butterﬂies that were seen removing polli-

nia of E. fulgens or with pollinia attached to the proboscis), (b)

potential pollinators (not seen removing pollinia of E. fulgens

but with the potential of being pollinators according to their

characteristics of behaviour, size, vision system and feeding

habits), and (c) not pollinators (not seen removing pollinia of

E. fulgens and no potential of being pollinators due to incom-

patible characteristics). Some of the butterﬂies interacted with

individuals of E. fulgens but did not remove pollinia (e.g. ovi-

posited), in which case they were not classiﬁed as ﬂower visi-

tors, but we indicate the presence of interaction in Fig. S5.

Plot experiment

The plot experiment was conducted using 60 E. fulgens individ-

uals cultivated in common garden conditions at the experi-

mental area of the Department of Plant Biology, Campus of

the Universidade Estadual de Campinas (Campinas –SP, Bra-

zil). To test whether the presence of different colour morphs

affects reproductive success, we assembled different plots of

cultivated individuals and exposed them to local wild pollina-

tors. Each plot contained ca. 20 inﬂorescences. Mono- and

polymorphic plots were placed far from each other (>20 m) in

an open area, where naturally occurring butterﬂies are present,

including those species already described as pollinators of E.

fulgens. We repeated the experiment 12 times, randomising

individuals and plot positions. Each round of exposure lasted

5 days, with a 9-day break between them. After each round,

MRS and FRS were estimated, as explained above.

In these plots, we estimated percentage MRS (related to pol-

linia removal; PR) and FRS (related to fruit formation; FF)

over time. We assembled three monomorphic plots (i.e. plots

with only Y, only O and only R colour morphs; hereafter, MY,

MO and MR) and one polymorphic plot (with Y, O and R

morphs in the same proportion; hereafter, P, and morphs

inside them PY, PO and PR). Percentage MRS and FRS were

calculated for each of the four plots (MRS

MY/MO/MR/P

and

FRS

MY/MO/MR/P

) and for each of the three colour morphs

within the polymorphic plot (MRS

PY/PO/PR

and FRS

PY/PO/PR

)

using the following formulae (here referring to the Y morph,

but similarly applied for all three morphs):

MRSMY =PRMY=PRMY þPRMO þPRMR þPRP

ðÞ100;

FRSMY =FFMY=FFMY þFFMO þFFMR þFFP

ðÞ100;

MRSPY =PRPY=PRPY þPRPO þPRPR

ðÞ100;

FRSPY =FFPY=FFPY þFFPO þFFPR

ðÞ100

To understand which species of Lepidoptera characterise

E. fulgens pollinator community at the experimental suburban

area, we performed direct observations (for a total of 10 h)

using the approach used for the natural populations survey.

Pollen limitation

To test for pollen limitation, in the BE population we per-

formed manual crosses in the ﬁeld, saturating 142 ﬂowers from

16 individuals with pollinia collected from other individuals of

the same population. On these individuals, we thus estimated

the number of ﬂowers pollinated and the number of fruits pro-

duced. We then compared these data with data collected in all

ten ﬁeld expeditions on open-pollinated individuals from the

same BE population. We calculated a pollen limitation index

for fruit formation using the following equation (PL; Larson &

Barrett 2000):

PL =1P

o=P

ðÞ

where P

is the proportion of fruits in open-pollinated plants,

and P

is the proportion of fruits in pollen-saturated plants.

The index ranges from 0 (no pollen limitation) to 1 (high pol-

len limitation).

Statistical analysis

To explore the level of phenotypic variability in E. fulgens,we

calculated a coefﬁcient of variation (CV) as the ratio between

standard deviation and mean for each of the eight investigated

ﬂoral traits in the two ﬂowering seasons then averaged them to

obtain a single value representative for the species.

Relative frequency and phenotypic differences across the

three ﬂower colour morphs were tested in the two populations

using Dunn Kruskal-Wallis multiple comparisons (with Bon-

ferroni correction) for pairwise comparisons. The same

approach was used to detect differences across the three colour

morphs MRS and FRS, both in the natural populations survey

and in the plot experiment. To detect a putative temporal vari-

ation in reproductive success, these parameters were also inves-

tigated separately in the two ﬂowering seasons and in three

different periods within one ﬂowering season. The

three periods were identiﬁed according to average precipitation

data from 1990 to 2022 for Campinas, provided by the Center

for Meteorological and Climatic Research Applied to Agricul-

ture of the Universidade Estadual de Campinas (https://www.

cpa.unicamp.br/), as: increasing rainfall (October to early

December, Period 1), elevated rainfall (mid-December to early

February, Period 2) and decreasing rainfall (mid-February to

end of March, Period 3).

To infer the strength and direction of selection in both

populations, in each colour morph we estimated selection gra-

dients in the two ﬂowering seasons using data collected on each

labelled individual, as described in the previous section. In BE

we also assessed differences in selection separately in each of

the three periods within the ﬂowering season. To do this, ﬁrst

we tested for the presence of intercorrelated ﬂoral traits using a

Spearman’s rank correlation test in order to remove highly cor-

related traits (>0.8). Then, following Lande & Arnold (1983),

we performed multiple single linear regressions using MRS and

FRS as response variables and standardised ﬂoral traits (z-

Plant Biology

Colour polymorphism in Epidendrum Arida, Pinheiro, Laccetti, Camargo, Freitas & Scopece

scores) as predictors. All the regression models were performed

using the lme4 R package (Bates et al.2015).

RESULTS

Flower phenotype polymorphism

Reﬂectance data showed different colour polymorphism pat-

terns on different ﬂoral parts: labellum showed continuous var-

iation, petal and lateral sepal showed discrete polymorphism,

and dorsal sepal showed overlap between orange and yellow

(Fig. 1B). Morphometric data revealed a moderately low CV in

E. fulgens, however CVs of different morphological traits

showed some differences (PetL =6.59%, PetW =13.76%,

LabA =14.46%, LatSepL =7.38%, LatSepW =7.50%,

DorSepL =7.30%, DorSepW =8.68%). LabS was mainly

explained by variation in PC1 (23.48% variance explained)

which represents the distance between the upper lobes of the

labellum (see Fig. S2). The three colour morphs showed signiﬁ-

cant differences in PetW, PetL, LatSepL and DorSepL in the BE

population (Figs. 2and 3), while in CA we found signiﬁcant

differences in LabS (Fig. 2, Fig. S3).

Natural populations survey and pollen limitation

The frequency of the three ﬂower colour morphs in natural

populations differed between BE and CA. In the BE popula-

tion, Y and R had the same frequency, and both were different

from O, which was the most frequent (Dunn’s test

YvsO

=4.56,

P<0.001; Dunn’s test

RvsO

=4.52, P<0.001), while in CA, the

three colour morphs differed from each other, with R being less

frequent, followed by Y, and Obeing most frequent (Dunn’s

test

RvsY

=1.92, P=0.02; Dunn’s test

RvsO

=3.90, P<0.001;

Dunn’s test

YvsO

=1.98, P=0.03) (see Fig. S1).

In both populations, the three morphs showed no signiﬁcant

differences in MRS and FRS in either of the two ﬂowering sea-

sons (Fig. 3). The same result was observed in the BE

population, by dividing the ﬂowering seasons in three different

periods (see Fig. S4).

Overall, the results obtained in the selection gradients analy-

sis suggest few marginally signiﬁcant values, indicating a weak

directional selection, with ﬂoral traits being under different

selection regimes in the two populations and in the two sea-

sons. In particular, during the ﬁrst season, in BE, there was a

signiﬁcant negative selection on PetW for MRS in the Ymorph,

a signiﬁcant negative selection on LabS for MRS in the O

morph, and a signiﬁcant positive selection on PetL for FRS in

the R morph. In the same population, in the second season,

there was a signiﬁcant positive selection on PetW for FRS, both

in the O and R morph (Table 1; Fig. 4A). Instead, in CA, in the

ﬁrst season, we detected a signiﬁcant positive selection on LabS

for FRS in the R morph. In the second season, there was a sig-

niﬁcant positive selection on PetW for MRS in the Y morph, a

signiﬁcant positive selection both for MRS and FRS on PetW

in the O morph, and a signiﬁcant positive selection on

PetW for FRS in the R morph (Table 2; Fig. 4B). In BE, by

dividing each season in different periods, different selective

patterns were found in the different periods (Table 3).

In the BE and CA populations, a total of 35 species of Lepi-

doptera were identiﬁed (see Fig. S5). Thirteen of these species

were observed pollinating E. fulgens or with pollinia attached

to their proboscis. In total, we observed 16 events of pollina-

tion (average 0.2 pollination events per hour of observation).

Of the 35 observed species, eight were only seen during the ﬁrst

ﬂowering season, 23 in the second season and four in both sea-

sons. In the BE population, pollen limitation was 0.82, with an

average proportion of fruits in open-pollinated individuals of

0.16 and in pollen-saturated plants of 0.91.

Plot experiment

In the plot experiment, there were no signiﬁcant differences for

any comparisons of FRS, possibly because of a very low fruiting

rate (average 0.013 fruits developed per ﬂower in the overall

Fig. 2. Phenotypic traits showing signiﬁcant differences among colour morphs of Epidendrum fulgens in Bertioga (A to D, green charts) and Cardoso (E, blue

chart) populations: petal length (A), lateral sepal length (B), dorsal sepal length (C), petal width (D) and labellum shape (E). Different letters indicate signiﬁcant

differences (P<0.05).

Plant Biology

Arida, Pinheiro, Laccetti, Camargo, Freitas & Scopece Colour polymorphism in Epidendrum

plot experiment), which is common in food-deceptive species.

Over the whole experiment, the average MRS of the monomor-

phic plots (M) was also not signiﬁcantly different from that of

the polymorphic plot (P) (Fig. 5A). However, when separately

considering the monomorphic plots (MY, MO, MR) and

morphs within the polymorphic plots (PY, PO, PR), plants in

Fig. 3. Male reproductive success (MRS) and female reproductive success (FRS) of the three colour morphs (Y, O, R) in the two ﬂowering seasons (1 and 2) in

the two natural populations: Bertioga (A, green charts) and Ilha do Cardoso (B, blue charts). No signiﬁcant differences (P<0.05) were detected.

Table 1. Selection gradients in the Bertioga population across the two ﬂowering seasons.

yellow orange red

1st ﬂowering

season

2nd ﬂowering

season

1st ﬂowering

season

2nd ﬂowering

season

1st ﬂowering

season

2nd ﬂowering

season

MRS

Petal length (mm) 0.019 0.006 0.014 0.016 0.008 0.049

Petal width (mm) 0.149*0.036 0.153 0.108 0.094 0.181

PC1 0.023 0.012 0.185*0.002 0.066 0.034

Labellum area (mm

)0.005 0.005 0.010 0.005 0.006 0.009

FRS

Petal length (mm) 0.099 0.064 0.064 0.023 0.226*0.015

Petal width (mm) 0.103 0.175 0.175 0.348*** 0.051 0.270**

PC1 0.024 0.127 0.127 0.091 0.064 0.070

Labellum area (mm

) 0.003 0.006 0.006 0.010 0.003 0.006

***P≤0.001, **0.001 <P≤0.01, *0.01 <P≤0.05.

Plant Biology

Colour polymorphism in Epidendrum Arida, Pinheiro, Laccetti, Camargo, Freitas & Scopece

the PY plot had a higher reproductive success than R morph

plants in both treatments (MRS

>MRS

, MRS

)orO

morph plants in the monomorphic plot (MRS

>MRS

)

(Fig. 5B). By dividing the plot experiment in three different

periods according to rainfall regime, we also found signiﬁcant

differences in MRS in the ﬁrst two periods, while in Period 3

Fig. 4. Signiﬁcant selection gradients, with mean of predicted values (line) and 95% conﬁdence interval (line shade), of the morphological traits on each col-

our morph (Y, O, R, represented by their respective colour): petal width (PetW), labellum shape (LabS) and petal length (PetL) in both populations, Bertioga (A,

green charts) and Ilha do Cardoso (B, blue charts) in the two ﬂowering seasons (1 and 2). Asterisks and their respective colours indicate a signiﬁcant difference

in the selection gradient of the corresponding ﬂower colour morph. ***P≤0.001, **0.001 <P≤0.01, *0.01 <P≤0.05.

Table 2. Selection gradients in the Cardoso population across the two ﬂowering seasons.

yellow orange red

1st ﬂowering

season

2nd ﬂowering

season

1st ﬂowering

season

2nd ﬂowering

season

1st ﬂowering

season

2nd ﬂowering

season

MRS

Petal length (mm) 0.132 0.092 0.084 0.104 0.239 0.151

Petal width (mm) 0.435 0.680*0.429 0.437*0.517 0.542

PC1 0.005 0.021 0.091 0.035 0.117 0.005

Labellum area

(mm

)

0.021 0.018 0.020 0.003 0.007 0.004

FRS

Petal length (mm) 0.220 0.128 0.178 0.084 0.209 0.097

Petal width (mm) 0.234 0.379 0.243 0.912** 0.109 0.421*

PC1 0.155 0.033 0.130 0.023 0.253*0.006

Labellum area

(mm

)

0.005 0.005 0.004 0.017 0.009 0.008

**0.001 <P≤0.01, *0.01 <P≤0.05.

Plant Biology

Arida, Pinheiro, Laccetti, Camargo, Freitas & Scopece Colour polymorphism in Epidendrum

there were no differences for any of the comparisons (Fig. 6).

In Period 1, PY was higher than R and O in both treatments

(MRS

>MRS

, MRS

), and in Period 2,

PY was higher than R in both treatments (MRS

>MRS

MRS

) and higher than the O morph in the monomorphic

plot (MRS

>MRS

) even without considering periods.

We identiﬁed 12 species of Lepidoptera in the experimental

suburban area, seven of which are also present in the natural

population of BE (Fig. S5). Of these, four taxa were identiﬁed

as pollinators of E. fulgens and, in total, we observed four

events of pollination (average 0.4 pollination events per hour

of observation). Despite habitat differences, the potentially pol-

linating species in natural populations and in the experimental

area were very similar, or equivalent species in terms of habitat

preference (open and sunny areas).

DISCUSSION

The origin of ﬂower colour polymorphism is highly debated

(Sapir et al.2021), ranging from weak selection or neutral pro-

cesses (Wang et al.2016; Jacquemyn & Brys 2020) to active,

divergent selection (Eckhart et al.2006; Newman et al.2012;

Bergamo et al.2016). Whatever the mechanism of origin, the

presence of polymorphism can have important consequences

on pollinator behaviour, thus inﬂuencing plant reproductive

success and selection patterns. Here, by surveying natural

populations of the colour polymorphic deceptive orchid spe-

cies E. fulgens, and by conducting a manipulative experiment,

we demonstrated that pollinator preferences for different

morphs changes in space, in ﬂowering seasons and in rainfall

periods within a season. We also demonstrated that selection

Table 3. Selection gradients in the Bertioga population across the three periods of each of the two ﬂowering seasons.

1st ﬂowering season 2nd ﬂowering season

yellow yellow

period 1 period 2 period 3 period 1 period 2 period 3

MRS MRS

Petal length (mm) 0.284 0.795 0.282 Petal length (mm) 0.411 0.306 0.187

Petal width (mm) 0.110 0.600 0.455 Petal width (mm) 0.120 0.107 0.312

PC1 0.245 0.480 0.358 PC1 0.286 0.249 0.562

Labellum area (mm

) 0.000 0.031 0.009 Labellum area (mm

)0.002 0.019 0.006

FRS FRS

Petal length (mm) 0.179 0.091 0.073 Petal length (mm) 0.156 0.173 0.031

Petal width (mm) 0.320 0.260 0.074 Petal width (mm) 0.252 0.220 0.34

PC1 0.145 0.192 0.145 PC1 0.136 0.047 0.243

Labellum area (mm

) 0.018 0.013 0.008 Labellum area (mm

) 0.004 0.002 0.006

orange orange

period 1 period 2 period 3 period 1 period 2 period 3

MRS MRS

Petal length (mm) 0.530 0.025 0.351 Petal length (mm) 0.919 0.341 0.346

Petal width (mm) 0.481 0.221 0.065 Petal width (mm) 0.738*0.942** 0.171

PC1 0.502 0.395 0.356 PC1 0.374 0.060 0.315

Labellum area (mm

) 0.021 0.043 0.012 Labellum area (mm

)0.038 0.066*0.079

FRS FRS

Petal length (mm) 0.039 0.345 0.160 Petal length (mm) 0.065 0.364 0.059

Petal width (mm) 0.684 0.042 0.243 Petal width (mm) 0.981** 0.920** 0.455*

PC1 0.217 0.039 0.345*PC1 0.362 0.225 0.052

Labellum area (mm

)0.026 0.011 0.003 Labellum area (mm

)0.012 0.069*0.009

red red

period 1 period 2 period 3 period 1 period 2 period 3

MRS MRS

Petal length (mm) 0.318 0.209 0.492 Petal length (mm) 0.672 0.063 0.261

Petal width (mm) 0.324 0.508 0.442*Petal width (mm) 0.556 0.104 0.063

PC1 0.171 0.349 0.000 PC1 0.081 0.025 0.290

Labellum area (mm

) 0.002 0.015 0.023 Labellum area (mm

) 0.025 0.000 0.000

FRS FRS

Petal length (mm) 0.982** 0.306 0.032 Petal length (mm) 0.845 0.089 0.011

Petal width (mm) 0.601 0.200 0.223 Petal width (mm) 0.932** 0.041 0.145

PC1 0.23 0.093 0.370*PC1 0.202 0.165 0.240

Labellum area (mm

) 0.019 0.002 0.015 Labellum area (mm

) 0.018 0.003 0.006

**0.001 <P≤0.01, *0.01 <P≤0.05.

Plant Biology

Colour polymorphism in Epidendrum Arida, Pinheiro, Laccetti, Camargo, Freitas & Scopece

on ﬂoral traits involved in pollinator attraction is weak but fol-

lowed different trajectories in different colour morphs and that

pollinators prefer one colour morph over the others. Taken

together, these results suggest that pollinators are able to detect

different ﬂower colour morphs, that different compositions of

the pollinator community change pollination patterns, and that

this ability has consequences for plant reproductive perfor-

mance and on the evolution of ﬂoral polymorphic traits.

The presence of a polymorphism in ﬂower colour in E. ful-

gens has been known since the ﬁrst botanical explorations in

the Neotropical region (Reichenbach 1878). Whether it was

continuous or discrete variation was, however, not tested.

Here, to resolve this issue, we analysed the reﬂectance spectrum

of different ﬂower parts of the extreme colour morphs (R and

Y) and of the intermediate O morph. Our results show a clear

discrete polymorphism in petals and lateral sepals, but not in

the labellum (Fig. 1). Overall, a discrete polymorphism in a

colour trait is often considered to result from genetic variation

at a single or a few loci involved in pigment biosynthesis (e.g.

Wu et al.2013). Although determination of the genetic archi-

tecture of this phenotypic trait is beyond the scopes of our

study, different phenotypic patterns in different ﬂoral parts

might point to a role for expression rather than inherited

genetic mutations. Alternatively, the presence of a modiﬁer

gene expressed in petals and lateral sepals (but not in the

labellum) could also alter expression of ﬂower colour genes

through its action (e.g. Scopece et al.2020).

Discrete ﬂower colour polymorphism is infrequent but phy-

logenetically widespread and is particularly common in decep-

tive orchids (e.g. Gigord et al.2001; Juillet et al.2010;

Jers´akov´aet al.2015; Narbona et al.2018; Jim´enez-L ´opez

et al.2020). This association between colour polymorphism

and deceptive orchids has been considered a consequence of

negative frequency-dependent selection, because the rarest

morph should have an advantage, it in being less recognisable

by pollinators (e.g. Gigord et al.2001). However, by comparing

MRS and FRS in the three colour morphs in natural popula-

tions we found no signiﬁcant differences (Fig. 3), thus suggest-

ing that no morph is advantaged in natural populations,

contrary to the negative frequency-dependent selection

hypothesis. This general ﬁnding was further conﬁrmed after

dividing the ﬂowering season in three different periods deﬁned

by rainfall dynamics.

Different colour morphs can be subjected to different selec-

tion pressures if the pollinator set involved in their pollination

is somewhat different and has different preferences. In natural

populations, selection might be related to different factors, but

in severely pollen-limited species the strength of selection is

considered to be mainly related to action of the pollinators

(Sletvold et al.2010; Sletvold &

Agren 2014). In our study we

Fig. 5. Male reproductive success (MRS) and female reproductive success (FRS) of monomorphic and polymorphic plots (A); MRS and FRS of each monomor-

phic plot (MY, MO and MR) and each morph within the polymorphic plot (PY, PO and PR) (B). Different letters indicate signiﬁcant differences (P<0.05).

Plant Biology

Arida, Pinheiro, Laccetti, Camargo, Freitas & Scopece Colour polymorphism in Epidendrum

found that reproductive success of E. fulgens is severely limited

and that overall selection on ﬂoral traits is weak but varies in

space, in seasons and in rainfall periods. This result, already

reported for Mediterranean deceptive orchids (Scopece

et al.2017), is likely linked to variations in the pollinator com-

munity, a trend common in generalist plant species where local

differences in prevalent pollinators can lead to local adaptation

(Frachon et al.2023). Accordingly, our pollinator observations

found that the pollination strategy of E. fulgens is highly gener-

alist, with at least 12 Lepidoptera species (i.e. 35.3% of the 34

reported Lepidoptera species; see Fig. S5) contributing to pol-

len transfer in the studied populations. This situation is typical

of generalised food-deceptive species (reviewed by Fantinato

et al.2017), including several Epidendrum species (reviewed in

Pinheiro & Cozzolino 2013).

If selection pressures are continuous and maintain the

same direction, they can lead to morphological differentiation

(Harder & Johnson 2009). Here, we found coherence between

morphological differentiation and the direction of selection

in some studied traits. In the population of BE, for instance,

the trait PetL was positively selected (for FRS, Fig. 4A) only

in the R morph, which also has signiﬁcantly longer petals

than the other two morphs (Fig. 2A); PetW was negatively

selected (for MRS, Fig. 4A) only in the Y morph, which has

signiﬁcantly thinner petals than the other two morphs

(Fig. 2D). These results suggest the presence of a somewhat

different pollinator set among the different morphs that

might lead to the observed morphological differentiation of

colour morphs. Spatial and temporal habitat heterogeneity is

one mechanism explaining the maintenance of polymor-

phisms within populations (Delph & Kelly 2014), and, in this

context, the diversity of the pollinator assemblage might be

interpreted as an important community feature enhancing

species variations. In fact, de Mattos et al.(2023) showed that

pollinator diversity and abundance not only contribute to

morphological variation of ﬂowers but also improve the habi-

tat suitability for E. fulgens, partially shaping its geographic

distribution.

We found evidence of variable direction of natural selec-

tion, with variation among years, among populations and

acting on different ﬂoral traits (Tables 1,2). Despite these

variations, there was no evidence of any trait being under

positive selection for one ﬂowering season and negative for

the next one, which suggests that different pollinators,

indeed, have different preferences and their long-term consis-

tency of choice affects the reproductive pattern of the colour

morphs, resulting in assortative mating. Assortative mating is

a known mechanism for maintenance of phenotypic varia-

tion (Kondrashov & Shpak 1998) and could explain the

coherence between morphological trait variation in opposite

directions among colour morphs. This could contribute to

explaining maintenance of the observed polymorphism in

Fig. 6. Male reproductive success (MRS) and female reproductive success (FRS) of each monomorphic plot (MY, MO and MR) and each morph within the poly-

morphic plot (PY, PO and PR) in the three measured periods. Different letters indicate signiﬁcant differences (P<0.05).

Plant Biology

Colour polymorphism in Epidendrum Arida, Pinheiro, Laccetti, Camargo, Freitas & Scopece

E. fulgens, regarding its rich and dynamic pollinator commu-

nity (see Fig. S5). Also, we only detected this pattern in the

multi-year study, thus we emphasise the importance of

long-term studies for evolutionary questions and call for

more investigations of seasonal variation on selection pat-

terns in deceptive orchid species.

In deceptive orchids, phenotypic polymorphism is thought

to be involved in decreased pollinator avoidance learning, thus

leading to predictions of higher reproductive success in poly-

morphic populations (Heinrich 1975). To test this prediction,

we performed a plot experiment, manipulating the presence of

different colour morphs. Contrary to the prediction, mono-

morphic and polymorphic plots experienced similar levels of

MRS and FRS. This ﬁnding conﬁrms previous ﬁndings

(reviewed by Juillet & Scopece (2010)) and challenges the role

of polymorphism in decreasing pollinator avoidance learning.

The plot experiment also showed that the Y morph in the

polymorphic plot (PY) was more attractive than the R morph,

both in monomorphic and within polymorphic plots (MR and

PR) (MRS in Fig. 5B). This could be linked to the perception

ability of Neotropical butterﬂies, as documented in higher

attraction in the yellow spectrum for several butterﬂy species

(Weiss & Papaj 2003;

Omura & Honda 2005 and references

therein). Recent studies have shown that yellow colour may be

interpreted by butterﬂies as a positive sign of the presence of

nectar, thus being preferred over other colours (Maharaj &

Bourne 2017; Santana et al.2022). This higher attractiveness of

the Y morph was not observed in natural populations (likely

because of variables such as relative abundance, heterogeneous

distribution, sharing pollinators with other species of local

ﬂora, proximity to other E. fulgens populations, and/or elevated

richness of pollinator community), but might contribute to

explain why the Y morph is more abundant than the R morph

in the CA population. However, why the higher attraction of

the Y morph does not translate into overall higher frequency

of plants with this phenotype in the natural populations is still

an open question.

The three colour morphs have different abundances in natu-

ral populations, with a clear prevalence of the O morph over

the R and Y morphs (see Fig. S1). The prevalence of the O

colour morph in natural populations is likely linked to inherit-

ability of this intermediate phenotype. Indeed, Pinheiro

et al.(2010,2016) report hybrids in the Epidendrum genus with

orange ﬂowers, which were generated by crosses between spe-

cies with yellow and red/pink ﬂowers. Alternatively, ﬂower col-

our morphs can also show differential tolerances to abiotic

factors (Warren & Mackenzie 2001; Arista et al.2017) related

to the association between ﬂoral pigments and protective ﬂavo-

noids and anthocyanins, which can be selected either for or

against in different habitats (Jim´enez-L ´opez et al.2020).

Indeed, the O and R morphs also contain red pigments in their

stems and leaves, contrasting with the pure green leaves and

stems present in the Y morph (F Pinheiro pers. obs.). Future

studies should investigate how ﬂower pigments inﬂuence phys-

iological responses of vegetative organs related to soil proper-

ties (Schemske & Bierzychudek 2007;Wuet al.2022), drought,

light or heat stress (Arista et al.2017; Costa et al.2023).

Flower colour polymorphism is a common phenomenon in

nature, and its maintenance has been a topic of much debate

(Sapir et al.2021). Taken together, our results suggest that

ﬂower colour polymorphism is maintained by a combination

of factors, including weak but varying pollinator-mediated

selection, assortative mating due to different pollinator prefer-

ences, and heritability of the intermediate phenotype. We

detected variations in pollinator preferences and selection

intensities across space, seasons and rainfall periods, indicating

a dynamic interplay between pollinators and ﬂower morphs

through space and time (Scopece et al.2017; Frachon

et al.2023). Our manipulative experiment further demon-

strated that the presence of different colour morphs did not

signiﬁcantly affect overall reproductive success, suggesting that

the observed polymorphism is likely maintained by other

mechanisms, excluding the occurrence of deterring pollinator

avoidance learning. While the different colour morphs did not

show differences in their overall reproductive success in the

ﬁeld, our plot experiment revealed that the Y morph was more

attractive to butterﬂies than the R morph. The fact that the

higher attractiveness of the Y morph does not translate into an

overall higher frequency of plants carrying this phenotype in

the ﬁeld deserves further studies, to investigate the role of ﬂoral

pigments in other plant physiological responses to abiotic vari-

ables, such as water, light and heat stress. Furthermore, other

biotic interactions, such as mycorrhizal (Burkle &

Zabinski 2023) and ant–plant associations (Ibarra-Isassi & Oli-

veira 2018) are essential data for studies seeking to understand

patterns of adaptation at small geographic scales. Considering

all contributing factors, a more comprehensive understanding

of the selection pressures shaping ﬂoral variation in hyperdi-

verse regions (such as the Neotropics) can be achieved.

AUTHOR CONTRIBUTIONS

BLA, AVLF, GS and FP planned and designed the research.

BLA performed the pollinator observations and the experimen-

tal approach. BLA and MGGC conducted the color analyses.

BLA, GS and LL analyzed the selection signs in the data. AVLF

identiﬁed pollinator species. BLA and LL performed morpho-

logical analyses. BLA wrote the manuscript. FP, LL, MGGC,

AVLF and GS carefully reviewed the manuscript.

ACKNOWLEDGEMENTS

We are grateful to many co-workers and friends who helped

with the ﬁeldwork: Celso Perez dos Santos, Edlley Max Pessoa

da Silva, Gabriel Pavan Sabino, Giovana Narezi Trotta, Iris

Dechiare Passos Ribeiro, Lucas Oliveira Mello, Raphael da

Silva, Welington Luis Sachetti Jr. Thanks to Sime˜

ao de Souza

Moraes and Gustavo Mattos Accacio for help in identifying

butterﬂy species. We thank members of the Laboratory of Evo-

lutionary Ecology and Genomics (LEEG), Universidade Esta-

dual de Campinas (UNICAMP), for helpful discussions and

comments regarding our results on the origins of ﬂower colour

polymorphisms. This work was supported by the Fundac

¸˜

ao de

Amparo `a Pesquisa do Estado de S˜

ao Paulo (FAPESP; grant n

2020/02150-3, 2021/03868–8) and FAPESP CBioClima

(2021/10639-5), additional grants from Fundo de Apoio ao

Ensino, Pesquisa e Extens˜

ao (FAEPEX –FUNCAMP) to FP,

and fellowships to BLA (FAPESP grants no 2021/10798-6 and

2023/01736-2), AVLF (CNPq productivity grant no

304291/2020–0) and FP (CNPq productivity grant no

302849/2021-1). This study was also ﬁnanced in part by the

Coordenac

¸˜

ao de Aperfeic

¸oamento de Pessoal de N´

ıvel Superior

Plant Biology

Arida, Pinheiro, Laccetti, Camargo, Freitas & Scopece Colour polymorphism in Epidendrum

–Brasil (CAPES) –Finance Code 001. GS was also funded

under the National Recovery and Resilience Plan (NRRP), Mis-

sion 4 Component 2 Investment 1.4 –Call for tender No. 3138

of 16 December 2021, rectiﬁed by Decree n. 3175 of 18 Decem-

ber 2021 of the Italian Ministry of University and Research

funded by the European Union –NextGenerationEU. LL was

funded in the framework of a Ph.D. scholarship in the ﬁeld of

sustainable development, ﬁnancially supported by INPS

(National Institute of Social Insurance).

SUPPORTING INFORMATION

Additional supporting information may be found online in the

Supporting Information section at the end of the article.

Fig. S1. Boxplots of the overall number of individuals of

each ﬂower colour morph (Y, O and R) found in an area of

100 m

in both natural populations: Bertioga (A, green chart)

and Ilha do Cardoso (B, blue chart). Different letters indicate

signiﬁcant differences (P<0.05).

Fig. S2. (A) PC1 eigenvectors associated with each landmark

or semi-landmark and their distortion. (B) Image of a labellum

with positioned landmarks and semi-landmarks. (C) Percent-

age variance of labellum shape explained by each principal

component (from PC1 to PC42). (D) PCA plot of labellum

shape of all ﬂowers collected from both populations (BE and

CA) (y-axis is PC2 and x-axis is PC1; beside the x-axis is a rep-

resentative image of the shape variation on each direction of

PC1, according to its eigenvectors).

Fig. S3. Phenotypic traits with no signiﬁcant differences

among colour morphs of E. fulgens in Bertioga (A to D, green

charts) and Ilha do Cardoso (E to K, blue charts) populations:

labellum shape (A), lateral sepal width (B and I), dorsal sepal

width (C and J), labellum area (D and H), petal length (E), lat-

eral sepal length (F), dorsal sepal length (G) and petal width (K).

Different letters indicate signiﬁcant differences (P<0.05).

Fig. S4. Male reproductive success (MRS) and female repro-

ductive success (FRS) of the three colour morphs (Y, O, R) in

the BE population after dividing the ﬂowering seasons in three

rainfall periods.

Fig. S5. Lepidoptera community in the two natural popula-

tions, Bertioga (BE) and Ilha do Cardoso (CA), and in the

experimental suburban area. Dots represent presence of the

respective butterﬂy species (empty dots for presence without

interaction; ﬁlled dots for presence with interaction with Epi-

dendrum fulgens), and line types represent their category: polli-

nators (full line), potential pollinators (dashed line), not

pollinators (dotted line).

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Putative Signals of Generalist Plant Species Adaptation to Local Pollinator Communities and Abiotic Factors

Article

Full-text available

Feb 2023
MOL BIOL EVOL

The reproductive success of flowering plants with generalized pollination systems is influenced by the interactions with a diverse pollinator community and abiotic factors. However, knowledge about the adaptative potential of plants to complex ecological networks and the underlying genetic mechanisms is still limited. Based on a pool-sequencing approach of 21 natural populations of Brassica incana in Southern Italy, we combined a genome-environmental association analysis with a genome scan for signals of population genomic differentiation to discover genetic variants associated with ecological variation. We identified genomic regions putatively involved in the adaptation of B. incana to the identity of local pollinator functional categories and pollinator community composition. Interestingly, we observed several shared candidate genes associated with long-tongue bees, soil texture, and temperature variation. We established a genomic map of potential generalist flowering plant local adaptation to complex biotic interactions, and the importance of considering multiple environmental factors to describe the adaptive landscape of plant populations.

Pollinator-Mediated Selection on Floral Traits of Primula tibetica Differs Between Sites With Different Soil Water Contents and Among Different Levels of Nutrient Availability

Article

Full-text available

Mar 2022

Abiotic environmental factors are predicted to affect plant traits and the intensity of plant-pollinator interactions. However, knowledge of their potential effects on pollinator-mediated selection on floral traits is still limited. We separately estimated the effects of soil water (two sites with different soil water contents) and N-P-K nutrient availability (different levels of nutrient addition) on pollinator-mediated selection on floral traits of Primula tibetica (an insect-pollinated perennial herbaceous species). Our results demonstrated that floral traits, plant reproductive success and pollinator-mediated selection on floral traits varied between sites with different soil water contents and among different levels of nutrient addition. The strength of pollinator-mediated selection was stronger at the site with low soil water content than at the site with high soil water content, and first decreased and then increased with increasing N-P-K nutrient addition. Our results support the hypothesis that abiotic environmental factors influence the importance of pollinators in shaping floral evolution.

Honest signalling and the billboard effect: how Heliconiid pollinators respond to the trichromatic colour changing Lantana camara L. (Verbenaceae)

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Full-text available

Mar 2017

Plants communicate with their pollinators through an astonishing range of signals that serve as either honest or deceptive cues which draw in and inform potential visitors of possible rewards. In wild type sweet sage, Lantana camara, floral colour signals were associated with nectar volume and sucrose concentration, and many pollinator taxa quickly learned to associate these varying colour signals with rewards. We tested the hypothesis that if sweet sage is employing a generalist pollinator strategy based on a trichromatic changing floral presentation system of honest rewards for pollinators, then the following predictions will be realized: 1) pre-change yellow flowers will be visited more frequently by pollinators than post change orange, or red flowers; 2) pre-change yellow flowers will produce higher quality and greater quantities of sucrose rewards than post-change orange, or red flowers; 3) inflorescences with higher ratios of rewarding flowers to unrewarding flowers are more attractive at short distances; and 4) inflorescences with a combination of pre-change rewarding and post-change rewarding and unrewarding flowers will act as a multi-coloured advertising billboard and as such be most attractive at long distances. We found corroboration for all of the aforementioned predictions. Thus, sweet sage evolved a generalized pollination visitation system based on honest signalling―of reward quantity and quality tied to colour changing visual signals acting in consort to produce a billboard that was easily perceived and deciphered. These resulted in high visitation rates by many different taxa of pollinators, thus contributing to higher individual plant fitness.

Salt tolerance in a neotropical orchid in the absence of local adaptation to salt spray

Article

Jul 2024
AM J BOT

Premise Salt tolerance has rarely been investigated regionally in the neotropics and even more rarely in Orchidaceae, one of the largest families. Therefore, investigating local adaptation to salt spray and its physiological basis in Epidendrum fulgens , a neotropical orchid species, brings important new insights. Methods We assessed the degree of salt tolerance in E. fulgens by testing whether coastal populations are more tolerant to salt, which could point to local adaptation. To understand the physiological basis of such salt tolerance, we exposed wild‐collected individuals to salt spray for 60 days, then measured leaf expansion, osmotic potential, sodium leaf concentration, chlorophyll leaf index, chlorophyll fluorescence, relative growth rate, and pressure–volume curves. Results There is no local adaptation to salt spray since both inland and coastal plants have a high tolerance to salt stress. This tolerance is explained by the ability to tolerate high concentrations of salt in leaf tissues, which is related to the high succulence displayed by this species. Conclusions We showed an unprecedented salt tolerance level for an orchid species, highlighting our limited knowledge of that trait beyond the traditional studied groups. Another interesting finding is that salt tolerance in E. fulgens is linked to succulence, is widespread, and is not the result of local adaptation. We suggest that E. fulgens and its allied species could be an interesting group to explore the evolution of important traits related to tolerance to salt stress, like succulence.

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Article

May 2023

Aim: The centre-periphery hypothesis (CPH) explains the decline of species abundance towards range limits and how this is driven by increasing ecological marginality. So far, most studies testing the CPH have focused on abiotic factors contributing to marginality, while the role of biotic interactions in limiting species distribution has been neglected. Here, we investigate both drivers' roles in restricting an orchid's range along a broad environmental gradient. Location: Atlantic Forest and Pampas grasslands (south and southeastern Brazil). Taxa: Sand dune orchid Epidendrum fulgens (Orchidaceae). Methods: We integrated empirical data on geographical distribution, pollinator richness and genetic diversity along the entire range of the species to investigate whether range limits match niche limits and whether habitat suitability declines towards lowand high-latitude species ranges. We performed niche models to predict niche limits and used polynomial and linear regression models to investigate the associations between ecological niche and species range as well as to test the relationship between genetic-derived metrics and the geographical and ecological distances. Results: Ecological conditions become more marginal towards the edges of the E. fulgens range, with an abrupt variation in precipitation. While pollinator richness increases habitat suitability of E. fulgens in the low-latitude edge range, climate has primarily shaped the species' high-latitude limit. Genetic diversity within populations decreases, while genetic differentiation increases towards both margins, although with a more consistent pattern for the low-latitudinal component. Main Conclusions: This study corroborates the predictions of CPH regarding ecological and genetic patterns of variation in space and highlights distinct factors limiting geographical distribution at the opposite margins of a latitudinal and narrowly distributed species. This improves our understanding on how biotic and abiotic variables limit species distribution ranges along latitudinal gradients in an extremely diverse and vulnerable tropical ecosystem, with potential for informing conservation practices.

Floral colour variation of Nicotiana glauca in native and non‐native ranges: Testing the role of pollinators' perception and abiotic factors

Article

Feb 2023
PLANT BIOLOGY

Invasive plants displaying disparate pollination environments and abiotic conditions in native and non‐native ranges provide ideal systems to test the role of different ecological factors driving flower colour variation. We quantified corolla reflectance of the ornithophilous South American Nicotiana glauca in native populations, where plants are pollinated by hummingbirds, and in populations from two invaded regions: South Africa, where plants are pollinated by sunbirds, and the Balearic island of Mallorca, where plants reproduce by selfing. Using visual modelling we examined how corolla reflectance could be perceived by floral visitors present in each region. Through Mantel tests we assessed a possible association between flower colour and different abiotic factors. Corolla reflectance variation (mainly along medium to long wavelengths, i.e. human green‐yellow to red colours) was greater among studied regions than within them. Flower colour was more similar between South America and South Africa, which share birds as pollinators. Within invaded regions, corolla reflectance variation was lower in South Africa, where populations could not be distinguished from each other by sunbirds, than in Spain, where populations could be distinguished from each other by their occasional visitors. Differences in corolla colour among populations were partially associated with differences in temperature. Our findings suggest that shifts in flower colour of N. glauca across native and invaded ranges could be shaped by changes in both pollination environment and climatic factors. This is the first study on plant invasions considering visual perception of different pollinators and abiotic drivers of flower colour variation.

Mycorrhizae influence plant vegetative and floral traits and intraspecific trait variation

Article

Nov 2022

Premise: Arbuscular mycorrhizal fungi (AMF) can strongly influence host plant vegetative growth, but less is known about AMF effects on other plant traits, the relative impacts of AMF on vegetative growth versus floral traits, or AMF-induced intraspecific variation in traits. Methods: In an experimental greenhouse study, we inoculated seven species of wildflowers with six species of AMF in a factorial design. We assessed how AMF-forb combinations influenced plant survival, vegetative biomass and floral traits, and whether AMF effects on floral traits were similar in magnitude and direction to effects on vegetative biomass. For one forb species, we investigated intraspecific plant trait variation within and across AMF treatments. Key results: AMF species varied in their effects on host plants, from negative to positive effects. AMF often had inconsistent effects on vegetative biomass versus floral traits, and therefore quantifying one or the other may provide a misleading representation of potential AMF effects. AMF treatments generated key variation in plant traits, especially floral traits, with potential consequences for plant-pollinator interactions. Given increased intraspecific trait variation in Linum lewisii plants across AMF species compared to uninoculated individuals or single AMF treatments, local AMF diversity and their host plant associations may scale up to influence community-wide patterns of trait variation and species interactions. Conclusions: These results have implications for predicting how aboveground communities are affected by belowground communities. Including AMF effects on not just host plant biomass but also functional traits and trait variation will deepen our understanding of community structure and function, including pollination. This article is protected by copyright. All rights reserved.

Retention of colour-changed flowers increases pollinator attraction to Lantana undulata inflorescences

Article

Sep 2022
FLORA

Many flowers change colour as they age or after pollination, which can have direct effects on pollinator behaviour affecting both pollination (on the plant side) and foraging efficiency (on the insect side). The retention of colour-changed flowers might increase plant attractiveness to pollinators over long distances, while directing pollinators at short range towards flowers that still present rewards and are sexually receptive. In this study, we used Lantana undulata Schrank (Verbenaceae), which presents yellow-centered and white flowers within an inflorescence, as a model plant species to study the influence of floral colour change on pollinator visitation. We first show that colour change from yellow to white occurs over a 24-hour period regardless of pollination, and is associated with reduced stigma receptivity and nectar reward availability. We then used pollinator visitor observations and experimental manipulations of inflorescence display patterns to investigate the effects of floral colour change on pollinator attraction, behavior and choices. Pollinators exhibited strong preference for visiting yellow-centered flowers, suggesting the colour change to white in older flowers is an honest signal directing pollinators to rewarding and receptive flowers within the inflorescence. In addition, the absence of white flowers on manipulated inflorescences significantly reduced their attractiveness to pollinators. Our results suggest that the retention of colour-changed white flowers is important to attract pollinators at long distances, while the yellow colour of young flowers guides pollinators to the sexually viable, rewarding flowers on inflorescences at short distances, maximizing both pollination and foraging efficiency.

Visual and olfactory sensory responses of the butterfly Papilio maackii during foraging and courtship

Article

Aug 2021

Colors and chemicals are the most important cues for butterflies to localize mating partners and food. In this study, we used artificial flowers with different colors and plastic butterfly wing mimics to separate the impact of colors from other cues on foraging and courtship of the butterfly Papilio maackii. We found that males relied more on visual cues during foraging and courtship, but females responded more to olfactory signals. Males visited artificial flowers more frequently than females. However, when honey was applied on artificial flowers, the frequency of female's flower visits increased and was much higher than that of male's, suggesting that females may have stronger olfactory than visual senses. During courtship, females were passive, whereas males were very active in chasing females and driving away other males. Similar to courtship among butterflies in a natural population, males showed recognition ability to butterfly wing mimics, indicating that males could accurately identify mates using colors only without any chemical cues. We also analyzed volatiles and identified five types of male‐specific chemicals while no female‐specific chemicals were identified, suggesting that females might have used these male‐specific volatiles to recognize and accepted males during courtship. Pollination of the plant Primula sieboldii mainly depends on P. maackii, and the plant has flowers with purple and mauve colors. Coincidently, P. maackii showed color preference to blue and purple, suggesting that the butterfly and plants might have coevolved during the long course of interactions.

What Maintains Flower Colour Variation within Populations?

Article

Mar 2021

Natural selection acts on phenotypic trait variation. Understanding the mechanisms that create and maintain trait variation is fundamental to understanding the breadth of diversity seen on Earth. Flower colour is among the most conspicuous and highly diverse traits in nature. Most flowering plant populations have uniform floral colours, but a minority exhibit within-population colour variation, either discrete (polymorphic) or continuous. Colour variation is commonly maintained by balancing selection through multiple pollinators, opposing selection regimes, or fluctuating selection. Variation can also be maintained by heterozygote advantage or frequency-dependent selection. Neutral processes, or a lack of selection, may maintain variation, although this remains largely untested. We suggest several prospective research directions that may provide insight into the evolutionary drivers of trait variation.

The consequences of flower colour polymorphism on the reproductive success of a neotropical deceptive orchid

Abstract

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