Local shifts in floral biotic interactions in habitat edges and their effect on quantity and quality of plant offspring
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Local shifts in floral biotic interactions in habitat edges and their effect on quantity and quality of plant offspring
Domenico Gargano 0 2
Giuseppe Fenu 1
Liliana Bernardo 0 2
Associate Editor: James F. Cahill
0 Present address: Museo di Storia Naturale della Calabria ed Orto Botanico dell'Universita della Calabria , loc. Polifunzionale, I-87036 Arcavacata di Rende , Italy
1 Centro Conservazione Biodiversita (CCB), Dipartimento di Scienze della Vita e dell'Ambiente, Universita degli Studi di Cagliari, Viale Sant'Ignazio da Laconi , 11-13, I-09123 Cagliari , Italy
2 Dipartimento di Biologia, Ecologia e Scienze della Terra dell'Universita della Calabria , Via P. Bucci, I-87036 Arcavacata di Rende , Italy
Spatial shifts in insect fauna due to ecological heterogeneity can severely constrain plant reproduction. Nonetheless, data showing effects of insect visit patterns and intensity of mutualistic and/or antagonistic plantinsect interactions on plant reproduction over structured ecological gradients remain scarce. We investigated how changes in flower-visitor abundance, identity and behaviour over a forest-open habitat gradient affect plant biotic interactions, and quantitative and qualitative fitness in the edge-specialist Dianthus balbisii. Composition and behaviour of the insects visiting flowers of D. balbisii strongly varied over the study gradient, influencing strength and patterns of plant biotic interactions (i.e. herbivory and pollination likelihood). Seed set comparison in free- and manually pollinated flowers suggested spatial variations in the extent of quantitative pollen limitation, which appeared more pronounced at the gradient extremes. Such variations were congruent to patterns of flower visit and plant biotic interactions. The analyses on seed and seedling viability evidenced that spatial variation in amount and type of pollinators, and frequency of herbivory affected qualitative fitness of D. balbisii by influencing selfing and outcrossing rates. Our work emphasizes the role of plant biotic interactions as a fine-scale mediator of plant fitness in ecotones, highlighting that optimal plant reproduction can take place into a restricted interval of the ecological gradients occurring at forest edges. Reducing the habitat complexity typical of such transition contexts can threat edge-adapted plants.
Dianthus; dichogamy; edge effect; inbreeding depression; plant mating systems; pollen limitation; self-fertilization
Plant biotic interactions (i.e. plant functional
relationships with mutualistic and antagonist organisms; PBIs
hereafter) are a key driver of population processes in
angiosperms, the largest group within the plant kingdom.
In flowering plants, PBIs are mainly founded on
associations with insects, which may act as pollinators or
(Schoonhoven et al. 2005; Harder and Barrett 2006)
VC The Authors 2017. 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 (http://creativecommons.org/
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As far as mutualistic relationships are concerned,
308 006 flowering plant species, accounting for 87.5 % of
the whole angiosperm diversity, show animal-mediated
(Ollerton et al. 2011)
. The relevance of
plant–pollinator interactions in preserving viable plant
populations is emphasized by historical connections
between insect and plant extinction patterns
. Indeed, inadequate pollen delivery (i.e.
pollen limitation) is recognized to limit plant fitness
(Aguilar et al. 2006; Ghazoul 2005; Knight et al. 2005)
and, then, to affect angiosperm evolution
. Pollen limitation (PL) can derive either from
quantitative or qualitative inadequateness of the pollen
reaching the stigma
(Wilcock and Neiland 2002)
. In the
first case, PL originates from insufficient pollen transfer,
which is typically due to the scarcity of pollinators
(Ashman et al. 2004). Instead, qualitative PL is due to a
poor quality of the pollen admixture deposited onto
(Amat et al. 2011; Arceo-Gomez and Ashman 2014)
Variations in abundance, composition and behaviour of
pollinating fauna may represent a primary source of both
quantitative and qualitative PL
(Gomez et al. 2010)
insect variations can rise from habitat changes
2001; Chalcoff et al. 2012)
, human disturbance (Brys and
Jacquemyn 2012) and local structuring of plant
(Zorn-Arnold and Howe 2007; Le Cadre et al. 2008)
Although much attention has traditionally paid to
plant–pollinator relationships, recent work highlights the
need of integrating studies on both plant–pollinator and
plant–herbivore interactions in investigations on PBIs
. Herbivory often exerts major
constraints on plant reproduction
(Cariveau et al. 2004;
Ashman and Penet 2007)
. For instance, florivory may alter
the efficiency of relationships between plants and
pollinators. This may increase PL in plant populations, due to the
reduced pollen transfer caused by pollen consumption
(Hargreaves et al. 2009; Harder and Aizen 2010)
, and to
herbivory-induced variations in plant traits involved in
pollinator attraction, like floral display
(Penet et al. 2009; So~ber
et al. 2010)
(Lucas-Barbosa et al. 2011, 2016)
Such interplay between mutualistic and antagonistic
relationships has also important evolutionary implications,
often resulting in selection conflicts on traits mediating
insect attraction or avoidance
(Gomez 2003, 2005)
Because habitat variations influence patterns of insect
presence, abundance and behaviour
Caruso et al. 2003)
, plant processes relying on PBIs can
show relevant spatial variations (Gomez et al. 2010).
These variations may be particularly evident in proximity
of habitat edges. Indeed, the switch between different
habitats generates transition zones with emergent
ecological properties recognized as ecotones
In landscapes subjected to a long-lasting human impact,
most of spatial ecological heterogeneity (and ecotones)
derives from the alteration of pristine habitats like
forests. This contributes to form a kaleidoscope of transition
habitats, which change in space and time depending on
local patterns of disturbance and vegetation dynamics
(Dovciak and Brown 2014)
. Studying ecological
responses at forest edges (i.e. edge effect) is a key topic in
landscape ecology and biodiversity conservation
(Forman 1995; Lopez-Barrera et al. 2007; Hadley and
. Indeed, understanding how ecological
processes vary near forest edges is crucial to evaluate how
landscape-level dynamics, such as fragmentation, affect
(Ries et al. 2004)
. Overall, species
interactions are retained one of the main mechanisms
influencing biodiversity patterns in habitat edges
(Ries et al.
. Accordingly, plant population processes
depending on PBIs (i.e. herbivory, pollination and dispersal) are
influenced by the proximity to forest edges
Armesto 2006; Lopez-Barrera et al. 2007)
. The extent of
such kind of edge effect depends on local levels of
(Sarlov-Herlin 2001; Lopez-Barrera
et al. 2007)
, and disturbance
(Bhattacharya et al. 2003;
Huang et al. 2009)
. Because the functioning of PBIs is
influenced by the size and spatial configuration of
habitat patches suitable for plant species
Verboven et al. 2014)
, the influence of habitat
heterogeneity on PBIs becomes more relevant in highly
fragmented landscapes (Hadley and Betts 2012).
Studying PBIs patterns in the proximity of forest edges
would improve plant conservation in complex ecological
(Hadley and Betts 2012)
, as those found in the
Mediterranean region. In this area, due to the
fragmentation of natural habitats, habitat edges occupy a
conspicuous portion of current landscapes, and are expected to
augment in the future, especially in lowlands
et al. 2007)
. Consequently, such contexts will represent
the theatre for evolutionary and conservation challenges
of an increasing number of plants. Nonetheless, the
management requirements of such anthropogenic ecotones
are often neglected. To date, there is a substantial
scarcity of field research investigating how small-scale habitat
variations affect PBIs (including plant–pollinator and
plant–herbivore interactions), and then plant reproductive
performance along structured ecotone gradients.
We evaluated the influence of ecological
heterogeneity on PBI-driven population processes in Dianthus
balbisii Ser., an edge-specialist inhabiting ecotones between
woody and open habitats in Mediterranean low lands.
Field work aimed to (i) identify potential pollinators and
herbivores of D. balbisii; (ii) evaluate the aptitude of
different pollinators versus intra- or inter-plant visits;
(iii) check for visit frequency variations along a gradient
of light intensity (assumed as a proxy of habitat
VC The Authors 2017
structure); (iv) perform floral manipulations to compare
the outcome of natural pollinations with that of forced
crossing and forced selfing along the studied ecological
gradient. Such data were used to ask the following
questions: (i) How did insect patterns change along the
studied gradient? (ii) Did variations of insect visit patterns
cause functional shifts in PBIs (i.e. pollination likelihood,
herbivory rate, amount and quality of pollen transfer)?
(iii) Did such shift in PBIs influence quantitative and
qualitative components of plant reproductive fitness along
the studied ecological gradient?
By answering such questions, we would provide basic
insights on the relationships between PBIs and plant
reproductive fitness in anthropogenic ecotones, an integral
component of complex landscapes.
Study species and study area
The perennial Dianthus balbisii Ser. has a woody stock
bearing up to 20 flowering stems ending with a
multiflowered head (2–20 flowers head); the corolla is
reddish, by distinguishing D. balbisii from the closely related
D. guliae Janka
(Peruzzi and Gargano 2006)
flowering season is from late May to late June. The distribution
range of D. balbisii embraces the Central Mediterranean
area, where this species occurs in hilly areas at the
border between woody and open xeric communities
. As in several congeneric species
, the plant is self-compatible, and shows a
protandrous hermaphroditism with the possible occurrence of
female flowers (i.e. gynomonoicism and gynodiocism).
Dianthus species have rather specialized pollination
syndromes, involving Lepidoptera as main pollinators
. Floral traits and architecture (i.e. reddish
flowers with short calix grouped in dense heads) suggest
that D. balbisii belongs to the carnations pollinated by
(Erhardt 1988; Block et al. 2006)
observations (i.e. daily flower monitoring) also support
that in this species most of pollinations occur during the
day (D. Gargano pers. obs.). In the study area (Calabria,
Southern Italy, N 39 330; E 16 220), D. balbisii occurs near
the border of forests dominated by deciduous oaks, and
in adjacent garrigue-like communities.
Sampling individuals and structuring the habitat gradient
The overall study population accounted approximatively
for 150 plants; these were rather uniformly distributed
over a forest ecotone ( 150 50 m) encompassing two
adjacent sites in a highly anthropized area. In spring
2014, 83 individuals of D. balbisii were permanently
marked before flowering by selecting plants that were at
least 5 m apart. From the whole sample, 40 plants were
used to collect data about pollination and herbivory. The
remaining 43 plants were employed in experimental
pollinations. In this way, possible interference of floral
manipulation procedures (e.g. bagging) with PBIs were
The ecotone structure was determined based on
measures of canopy closure. This parameter was chosen
because variations in habitat canopy are recognized to
influence both abiotic (i.e. temperature and soil
(Joffre and Rambal 1993)
Chacon and Armesto 2006; Lopez-Barrera et al. 2007)
drivers of plant fitness. The position over the ecotone of
each sampled plant was determined by using light
intensity (LI) as a proxy of habitat canopy closure. A direct
measure of incident light was chosen because it can be
effective in recording small-scale canopy variations near
the ground surface, as congruent with the size of the
study species. Direct solar radiation measures can also
help in interpreting patterns of pollinator activity
(Kastinger and Weber 2001)
. Moreover, this method
allows many replications to obtain more stable average
values. LI was measured on the surface of the flower
heads by a luxometer (Panlux electronic 2, Gossen,
Nu¨ rnberg). For plants that had up to three flowering
stems the light intensity was measured on all stems, and
the resulting values were averaged. Instead, for plants
with many flowering stems, three measures were taken
on the three tallest stems, and the recorded values were
then averaged. All measures were carried out after the
canopy of deciduous trees completed its development.
To reduce the effect due to the moment in which they
were taken, all the measurements were repeated during
two consecutive sunny days (12–13 June) from 12.00
a.m. to 2.00 p.m.
LI measurements were used to structure a gradient of
light intensity encompassing five classes hosting a
comparable amount of sampled individuals: (i) LI < 500 Klux
(No. plants ¼ 16), (ii) 500 < LI < 1500 Klux (No. plants ¼
17), (iii) 1500 < LI < 2500 Klux (No. plants ¼ 17), (iv)
2500 < LI < 4000 Klux (No. plants ¼ 17), (v) LI > 4000
Klux (No. plants ¼ 16). Because canopy variations
regulate spatial resource patterns in temperate habitats
(Joffre and Rambal 1993)
, plants occurring in the same
LI interval were assumed to have a comparable amount
of available resources as they occurred under similar
Individual patch size (no. of stems, no. of buds per stem
and per plant) was estimated for the 40 plants (8 LI
class) selected to monitor insect visits.
VC The Authors 2017
Observations on insects visiting D. balbisii were carried
out over four consecutive days (from 12 June to 16 June)
in the middle of the species’ blooming season. Overall,
they accounted for 560 min of direct field observations
for recording each insect visit on each marked plant.
Such observations were organized in trials of 20 min,
which were carried out during fixed intervals in the
morning (from 10.30 a.m. to 12.30 a.m.) and in the
afternoon (from 3.00 p.m. to 5.00 p.m.). Plants were subjected
to 28 observation runs (16 in the morning and 12 in the
afternoon). To minimize disturbance to insects, the
observers remained seated some meter apart the plants.
Due to the gradual blooming typical of the study species,
each observation trial was preceded by the annotation
of the amount of flowers opened on the plants. This
allowed us to relate visit patterns to the effective flower
display (EFD ¼ no. of opened flowers on the plant at the
When possible, the identity of insect taxa was
assessed in the field. Unknown insects visiting D. balbisii
flowers were identified in laboratory with the help of
expert entomologists based on images and specimens
collected in the field. Taxa unidentified at the specific level
were assigned to higher taxonomic ranks (i.e. genus and
family). The observed insects were categorized in four
functional categories. Firstly, they were distinguished
into pollinators or herbivores based on literature
knowledge and field observations. Butterflies were always
considered as pollinators as they are the main pollinators of
. Also Bombylius major L.
(Diptera), that frequently visited flowers of D. balbisii,
was included among pollinators. Indeed, bee-flies are
recognized as pollinators in many plant species with
(Kastinger and Weber 2001)
. Then, the
primary pollinators (the lepidopteran Thymelicus sylvestris
Poda and B. major) were separated from secondary
pollinators (other lepidopterans, OTH) based on visit
frequency. Furthermore, T. sylvestris and B. major were
assigned to two distinct categories (named, respectively,
THY and BOM) because of relevant functional differences
(i.e. diverse preferences with respect to the investigated
gradient, diverse aptitude to within- and among-plant
visit). Finally, weevils and ants were classified as
(McCall and Irwin 2006; Penet et al. 2009)
and nectar robbers (Soper Gorden and Adler 2016),
respectively. Since weevils were widely dominant, all
florivores were included into a single category (HER). HER
also included pre-dispersal predators that were
identified in laboratory based on the caterpillars found in fruits
of D. balbisii collected in the field. Joining florivores and
pre-dispersal predators into a single unit was justified by
the very low amount of seeds produced by flowers that
experienced florivory (see below).
To estimate the potential for cross- and
selffertilization of each visiting taxon based on its
behavioural aptitude, visits were classified as (C) (visits among
individuals, potentially allowing for cross-pollination), or
(G) (visits within individual, potentially allowing for
geitonogamy). The (C) tag was used when insects visited an
individual of D. balbisii and then moved away.
Differently, the (G) tag applied to visits of insects coming
from other flowers of the same plant (in other words,
multiple consecutive visits on a same plant qualified
The 40 plants were monitored daily over 3 weeks (from
14 June to 5 July), by annotating amount and sex of
open flowers. Since the species is protandrous, the
hermaphrodite flowers were distinguished into males (i.e.
flowers bearing only emergent stamens), and
hermaphrodites (i.e. flowers bearing both stamens and stigma
branches). Finally, male-sterile flowers (i.e. bearing only
stigma branches) were classified as female. Flower sex
data were used: (i) to quantify the frequency of
malesterility within the population, and (ii) to estimate
pollination frequency over the study gradient (i.e. in
proterandrous species, large accumulations of flowers in the
hermaphrodite stage can indicate slow pollination
During such field trials we also recorded flowers that
resulted pollinated (i.e. flowers with closed corolla) or
showed signs of florivory (i.e. petals, stamens and/or
stigma branches eaten by herbivores). This work
consisted in 2737 field observations involving 656 flowers.
Fertility of perennial plants can be affected by several
maternal traits (e.g. age, size, history, genetics and
environmental conditions). This can bias fitness comparisons
among pollination treatments carried out on different
individuals. To reduce such bias, floral manipulations were
designed to obtain matched offspring for various
pollination protocols (see below) from each experimental plant.
To this end, from the initial 43 individuals, plants bearing
<5 flowers were not manipulated, and the final amount
of treated plants became 30 (6 LI class).
The pollination experiments performed on these
plants involved at least one flower for each of four
treatments: forced crossing (No. ¼ 80), forced selfing
(No. ¼ 76), autonomous selfing (No. ¼ 74) and free
pollination (No. ¼ 80). Except for the sample used for free
pollination, the flowers were isolated at the early bud stage
by bagging 1–2 inflorescences per plant. In flowers used
for forced cross-pollination, anthers were gently
VC The Authors 2017
removed with a dissection forceps before the emergence
of stigma to avoid occasional selfing. Hand-pollinations
(i.e. forced crossing and forced selfing) were carried out
by brushing one dehiscent anther per stigma branch; this
would allow for fertilizing all ovules (<100) usually found
in the ovaries of D. balbisii. The stigma was retained
receptive when it was well developed and appeared
glutinous and papillose. Self-pollen was taken from other
flowers of the same plant. Pollen used in forced-crossing
was taken from >30 plants, each distant >5 m from the
recipient plant. Occurrence and extent of autonomous
self-pollination was evaluated by leaving untreated 1–2
bagged flowers per plant. As the last flower in the head
closed petals or withered, the bag was removed to allow
fruit development under natural conditions.
All flowers left free were used to investigate rates of
herbivory, pollination and seed set under natural pollination
regime. At the end of the blooming season all fruits
occurring on the marked plants were collected. In the
laboratory, fruits were checked for occurrence of
predispersal predation (e.g. presence of caterpillars,
remnants of seeds eaten by herbivores). In these way fruits
were distinguished into three categories: predated,
aborted and fertilized. This screening accounted for 1185
fruits from 40 plants. Except for three cases (on 571), no
seeds were produced by flowers that experienced
florivory or pre-dispersal predation. Therefore, we annotated
for each flower the occurrence of herbivory or
pollination, and in the latter case the number of seeds.
The seeds contained in each fruit were counted and,
then, a sample of 450 seeds was germinated to assess
seed viability and early seedling survival rate. At the
beginning of the autumn seeds were sowed on wet paper in
Petri dishes at 18 C. The resulting seedlings were
transferred into 4 4 cm pots containing a mixture of peat
moss and compost with presence of pumice fragments.
Subsequently, 30-day old seedlings were transplanted
into 20 17 cm pots filled with brown soil and subjected
to periodical irrigation and fertilization (20 ml of a
mixture of ammonium (6 %), phosphorous (5 %), and
potassium (7 %) diluted in H2O was monthly supplied). The
plants were maintained in glasshouse for 6 months at
Botanic Garden of the Universita della Calabria.
The efficacy of the sampling effort in capturing
information on insects visiting D. balbisii was evaluated by a
rarefaction procedure on sample-based abundance data
(Colwell et al. 2012)
. To this end it was applied the
rarefaction function provided by EstimateS 9.1.0
The relationships between visit frequency (no. of
visits plant patch observation run) and EFD were
checked by a Pearson correlation test. Variations of visit
frequency across levels of light intensity were checked
by a Generalized Linear Model (GLM), by considering the
LI class as fixed factor and EFD as covariate. The
association between visitor type and light intensity, and
between visitor type and visit type was evaluated by
cross-tabulation analyses using chi-square as a measure
Variations of individual patch size (i.e. no. of stems per
plant, no. of buds per plant and no. of buds per stem)
across intervals of light intensity were analysed by
oneway Anova. Patterns of sex ratio were evaluated by
calculating from daily data the individual hermaphroditic
rate (no. of hermaphrodite flowers/total no. of open
flowers per plant) and the individual female rate (no. of
female flowers/total no. of open flowers per plant). Then,
differences among intervals of light intensity in average
individual hermaphroditic and female rates were
checked by one-way Anova.
The strength of PBIs was evaluated based on the
variations of herbivory, pollination chance and seed
set over the five LI classes. In the analyses, herbivory
accounted for both florivory and pre-dispersal
predation, because they had an equivalent effect of the
reproduction of D. balbisii (i.e. both caused absence of
seed-set). To include the effect of plant traits involved
in insect attraction on herbivory and pollination
likelihood, we comprised in such analyses the inflorescence
size (i.e. the amount of flowers occurring in the same
head of each studied fruit). Herbivory was a binary
variable indicating presence or absence of florivory and
pre-dispersal predation. The pollination chance also
consisted in binary data indicating occurrence or
absence (caused by lack of visits, herbivory or abortion)
of fertilization. Variations of herbivory and pollination
chance among LI classes were then analysed by GLM,
including the LI class as fixed factor and the
inflorescence size as covariate. Quantitative fitness analyses
were based on seed set (i.e. no. of seeds per fruit)
comparisons between naturally pollinated and
manipulated plants. Flowers that were not pollinated were
considered by assigning a seed set ¼ 0. Seed set in
notpredated flowers (570 flowers from 40 plants) was
analysed by one-way Anova to estimate seed set
variations among the 5 LI classes. To evaluate the effect of
qualitative pollen limitation on seed production, a GLM
was used to analyse the seed set of 75 fruits per each
of three treatments: forced-crossing, forced selfing,
natural pollination (3 fruits 5 plants 5 LI classes
3 treatments) using LI class and pollination treatment
as fixed factors.
VC The Authors 2017
Further qualitative fitness components (i.e. seed
germination and survival in 6-month-old seedlings) were
estimated by considering the sample of 450 seeds (6 seeds
5 plants 5 LI classes 3 pollination treatments)
obtained by plants under free-pollination, forced crossing
and forced selfing. Autonomous self-fertilization was not
considered because it was ineffective in most of cases.
Seed germination and seedling survival were treated as
binary variables, and were analysed by GLM (using LI
interval and pollination treatment as fixed factors). GLM
analyses on binary variables were performed by using
the LOGIT link function. Response variables accounting
for count data (i.e. visit frequency, no. seeds fruit) were
not normally distributed. Therefore, in GLM these
variables were analysed by using a LOG link function, which
is appropriate for data represented by non-negative
integer values. Because floral manipulations (i.e. pollen
supplementation) can promote resource reallocation within
plants, this may result in a higher pollen limitation in
free-pollinated flowers of manipulated plants
et al. 2006; Wesselingh 2007)
. The possible effects of
such reallocation processes were tested by a t-test on
independent samples on the seed set of 160 free
pollinated flowers, 80 were randomly selected from the 30
manipulated plants, and 80 from 30 non-manipulated
individuals within the same LI interval.
All these analyses were performed in SPSS 24 for
Windows (SPSS, Chicago, IL, USA).
Patterns of visit abundance, visitor type and behaviour over the studied gradient (question 1)
In average, we recorded 4.7 6 1.9 insect taxa per
observation run. The rarefaction function suggested that the
observation effort provided a reliable estimation of the insect
fauna interacting with the flowers of D. balbisii (Fig. 1).
GLM analyses indicated that light intensity influenced
visit frequency (Wald chi2 ¼ 29.46, P < 0.001). More than
71 % of documented visits occurred under a light
intensity exceeding 2500 Klux (Fig. 2A). The effective floral
display (EFD) also significantly influenced the visit
frequency (Wald chi2 ¼ 24.05, P < 0.001). Accordingly,
visit frequency was significantly related to EFD (r ¼ 0.389;
P < 0.001; No. ¼ 384). However, the significant
interaction between LI and EFD (Wald chi2 ¼ 15.89, P ¼ 0.003)
suggested that the effect of EFD on visit frequency varied
along the ecological gradient.
The list and frequency of recognized insects, along with
their putative functional role, is provided in Table S1 [see
Supporting Information]. Among pollinators, T. sylvestris
(THY) was responsible for 53.2 % of visits, followed by B.
major (BOM ¼ 16.9 %), and a mixture of other butterflies
(OTH ¼ 5.6 %). The remaining visits were due to herbivores
(HER ¼ 24.3 %). Among the herbivores, florivores (85.5 %)
were much more frequent than pollen/nectar eaters
(14.5 %) [see Supporting Information—Table S1]. The
frequency of the different visitors varied among intervals of
light intensity (Wald chi2 ¼ 86.389, P < 0.001). The relative
visit frequency of THY ranged between 0.28 (LI < 1000
Klux) and 0.73 (2500 < LI < 3500 Klux), and then
decreased at higher light intensities (Fig. 2B). Instead, BOM
was the most frequent visitor in the lowest LI class (Fig.
2B). The proportion of visits due to HER varied from 0.08 to
0.36, with lower frequencies at intermediate LI intervals
(Fig. 2B). The remaining putative pollinators had much
lower visit frequency (<0.1 in most LI intervals) (Fig. 2B).
Different visitors were significantly associated to
different movement patterns (i.e. among- and within-plant)
(Wald chi2 ¼ 18.34, P < 0.001). Most of within-plant
movements were due to BOM (50.7 %) and HER (69.7 %).
Instead, movements among plants prevailed in OTH
(60.9 %) and, especially, in THY (76.0 %).
Relating insect visit and flowering regime to patterns of PBIs (question 2)
Patch size traits differed among intervals of light intensity
(Table 1). The no. of buds per plant (F ¼ 4.948, P < 0.001;
N ¼ 40), the no. of stems per plant (F ¼ 3.454, P ¼ 0.02, N
¼ 40), and the no. of buds per stem (F ¼ 4.321, P < 0.001,
N ¼ 40) significantly varied over the gradient, reaching
higher values in sunny places. The average individual
amount of open flowers found in daily recognitions
decreased toward high LI intervals (Fig. 3A), while the rate of
florivory showed an opposite patterns (Fig. 3A). Daily
average values of individual hermaphroditic rate strongly
differed over the LI gradient (F ¼ 15.117, P < 0.001, N ¼ 40),
revealing larger scores at lower LI (Fig. 3B). This suggested
that, at lower LI, flowers required a longer time to be
visited, as congruent with general patterns of insect visit. On
the contrary, individual female rate (Fig. 3B) did not reveal
significant patterns (F ¼ 0.167, P ¼ 0.955, N ¼ 40).
A variation of PBIs along the studied gradient was
confirmed by GLM. Indeed, light intensity, inflorescence size
and their interaction) had significant effects on herbivory
and pollination chance (Table 2). Pollination increased
from low to intermediate intervals of light intensity and
then decreased at higher LI, while herbivory showed an
opposite pattern (Fig. 4).
Quantitative and qualitative consequences on plant fitness (question 3)
The no. of seeds per fruit varied significantly over the
gradient (F ¼ 14.439, P < 0.001), showing a decline in higher
LI intervals (Fig. 5A).
VC The Authors 2017
Among manipulated plants, 25 individuals (5 LI class)
produced matched fruits for forced cross-pollination,
forced self-pollination and free-pollination. Instead, many
flowers and plants failed to produce seeds by
autonomous selfing (<14 % of flowers produced fruits
containing in average 2.45 6 6.88 seeds). The seed-set of
freepollinated flowers on non-manipulated plants did not
differ from free-pollinated flowers on plants subjected to
experimental pollinations (t ¼ 1.542, df ¼ 160, P ¼ 0.098).
The two way ANOVA on manipulated flowers evidenced
substantial differences in seed set among forced selfing,
forced crossing and free pollination (Table 3). The lower
seed set was found in flowers left to free pollination. In
14 % of cases they did not set fruits, and their average
seed set (14.75 6 7.78) was lower than those found in
flowers treated with forced selfing (18.05 6 6.56) and
forced crossing (23.41 6 10.29). LI had significant effect
on seed set patterns also in experimentally pollinated
flowers (Table 3). The no. of seeds obtained by free
pollination was lower than in flowers treated with
forced crossing (average difference ¼ 0 8.66, P < 0.001,
N ¼ 150) and forced selfing (average difference ¼ 0 2.79,
P ¼ 0.003, N ¼ 150). As shown in Fig. 5A, seed set from
free pollination overcame forced selfing in two LI intervals
(LI3: difference ¼ 6.23, P < 0.001, N ¼ 30; LI4:
difference ¼ 3.21, P ¼ 0.03, N ¼ 30). In the same LI intervals,
the difference between free pollination and hand
crosspollination remained significant but it was less
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LI class Stems 3 plant Buds 3 plant Buds 3 stem EFD
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Source of variation F P
Light intensity 26.489 <0.001
higher than variations in germination (Fig. 5B, C). Overall,
germination rate in cross-pollinated flowers was
significantly higher to both free pollinated (83.3 % vs. 72.0 %,
P ¼ 0.024) and self-pollinated ones (83.3 % vs. 66.0 %,
P ¼ 0.001). On the contrary, the difference between free
pollination and forced selfing was not statistically
significant (P ¼ 0.231). Seedling survival revealed a similar
pattern, although the amount of survived plants was
generally lower compared to germination rates
(crosspollination ¼ 75.3 %; free-pollination ¼ 64.7 %;
self-pollination ¼ 52.0 %). The differences between
crosspollination and forced selfing (P < 0.001) were higher
than between cross-pollination and free pollination
(P ¼ 0.048). Instead, differences between free pollination
and forced self-pollination were marginally significant
(P ¼ 0.051). At intermediate LI intervals (LI ¼ 3–4), fitness
differences between cross- and free-pollinations were
not significant (P ¼ 0.456 and P ¼ 0.527 for germination
and survival, respectively) (Fig. 5B, C). Instead, at highest
LI the fitness related to free pollination approached that
of self-pollination for both germination (P ¼ 0.229) and
survival (P ¼ 0.171) (Fig. 5B, C).
Linking ecological heterogeneity to patterns of visit frequency and type (question 1)
Spatial ecological heterogeneity can promote substantial
small-scale effects on insect webs
(Totland 2001; Caruso
et al. 2003)
. Accordingly, our data evidenced substantial
variation in the overall and relative frequency of insect
visits over the investigated ecological gradient. The
individuals of D. balbisii occurring in sunny places
experienced high visitation rate, confirming the general
expectation that, in forest-edge contexts, insects prefer
the more productive open patches (Chacon and Armesto
2006). However, we also recorded relevant variation in
the relative visit rate of different insects over the
investigated gradient. Substantially, this resulted in three major
patterns: (i) infrequent visits that were mainly due to
Bombylius flies at low LI, (ii) high visitation rate,
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prevalently due to butterflies, at intermediate LI and (iii)
increasing visits of florivores at higher LI.
Linking ecological heterogeneity and PBIs patterns (question 2)
The shifts in the visitation rate of mutualistic and
antagonist insects influenced strength and patterns of PBIs over
the LI gradient. Indeed, the individual patterns of flower
accumulation showed a lower visitation rate at low LI.
Instead, a prevalence of mutualistic (i.e. pollination) and
antagonist (i.e. herbivory) interactions occurred at
intermediate and high LI, respectively. Because the interplay
between mutualistic and antagonistic interactions can
generate selection conflicts on plant traits involved in
(Gomez 2003, 2008)
, such patterns of insect
preferences could have affected the relationships between
floral display size and pollinators. Indeed, although larger
plant patches occurred at higher LI, here herbivory
overcame pollination (Fig. 4). The frequency of herbivores at
high LI, and their aptitude to perform multiple within-plant
visits may have constrained pollination in many-flowered
plants by reducing the available flowers, and by limiting
the plant ability in attracting pollinators. Accordingly,
herbivory was often found to alter plant traits involved in
pollinator attraction, like scent
(Lucas-Barbosa et al. 2011,
and floral display
(Penet et al. 2009; So~ber et al.
. Such findings agree with a non-additive pattern
between positive and negative interactions, highlighting that
a large floral display size can become disadvantageous in
(Gomez 2003, 2005)
Linking ecological heterogeneity, PBIs and plant fitness (question 3)
Hand-pollinations may bias evaluation of pollen
limitation, by promoting resource reallocation between
non-manipulated and manipulated flowers of treated
(Knight et al. 2006; Wesselingh 2007)
. In our case,
the seed-set of free-pollinated flowers from
nonmanipulated and manipulated plants was comparable.
This suggested that: (i) the effect of within-plant
reallocation processes promoted by pollen supplementation
was negligible, and (ii) the presence of bags on
experimental plants did not influence pollinators.
Seed-set comparison between naturally and
experimentally pollinated flowers suggested a significant pollen
limitation in the studied population. Pollen limitation is a
frequent consequence of human disturbance
(Eckert et al.
2010; Brys and Jacquemyn 2012)
, but in our case it
seemed linked to the variations of insect activity occurred
over the studied ecological gradient. In addition, as
common in Dianthus
(Jennersten 1987; Collin and Shykoff
2003; Gargano et al. 2009)
, D. balbisii showed a little ability
for autonomous self-fertilization, and this can have
favored the rise of pollen limitation. Indeed, functional
limitation for self-fertilization limit the chances of
reproductive assurance (Kalisz et al. 2004). Anyway, the
extent of pollen limitation varied greatly in our system,
being higher at the gradient extremes. Variations in insect
assemblages were demonstrated to generate spatial
patterns of pollen limitation
(Gomez et al. 2010)
our data evidenced that spatial structuring of pollen
limitation can occur at very small scale, following the
ecological gradients typical of transition habitats. Although
pollinators are essential to allow pollen transfer
et al. 2004)
, herbivory can also influence pollen delivery
rates. Florivory can reduce the pollen available to
(Hargreaves et al. 2009; Harder and Aizen 2010)
limit the efficiency of plant–pollinator interactions
et al. 2009; So~ber et al. 2010; Lucas-Barbosa et al. 2011,
. Accordingly, the variations of both pollinators and
herbivores contributed to model patterns of pollen
limitation over the investigated gradient.
Pollen limitation also involves qualitative components
of plant reproduction (i.e. offspring viability), which depend
on the features of transferred pollen
(Wilcock and Neiland
2002; Amat et al. 2011; Arceo-Gomez and Ashman 2014)
The interplay between the structuring of plant populations
and insect assemblages can affect the quality of pollen
delivery (i.e. cross- vs. self-pollen) and, then, the viability in
the subsequent offspring
(Zorn-Arnold and Howe 2007; Le
Cadre et al. 2008)
. In our study system, viability differed
between progenies from hand-crosspollination and free
pollination at low and high LI (Fig. 5B, C). Although
Dianthus species are fully self-compatible, the presence of
protandry and male-sterility promotes high outcrossing
rates in their populations
(Collin and Shykoff 2003)
consequent genetic load can result in severe inbreeding
depression under enhanced selfing rates
(Collin et al.
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2009; Gargano et al. 2009, 2011, 2015)
. Therefore, the
observed fitness variations can result from local variations in
relative amount of cross- and self-pollinations. At
intermediate LI, the fitness of naturally produced offspring was
roughly equidistant from forced crossing and forced
selfing (Fig. 5B, C). This would be compatible with the mixture
of self- and cross-pollination typically occurring in plant
(Wilcock and Neiland 2002)
. Instead, toward
the gradient extremes the fitness of progenies from
freepollinated flowers declined, approaching the values
detected after forced selfing (Fig. 5B, C). This suggested
increased selfing rates. The accumulation of flowers in
hermaphrodite phase on individuals, and the multiple
withinplant visits due to Bombylius could have promoted
inbreeding at low LI. Indeed, the presence of many open
flowers on a single plant diminishes the effectiveness of
dichogamy in preventing self-fertilization via geitonogamy
(Petterson 1992; Hidalgo and Hubera 2001)
. Such a
limitation can be exacerbated if the prevalent pollinators travel
(Zorn-Arnold and Howe 2007)
Instead, herbivory could have enhanced selfing rates at
high LI. Indeed, by reducing flower display and plant
attractiveness, massive florivory can increases the chances
of self-fertilization due to sporadic pollinator visits
et al. 2009; So~ber et al. 2010)
. Then, the observed patterns
of offspring viability can have reflected varying rates of
inbreeding promoted by the interplay of: (i) habitat
heterogeneity, (ii) insect fauna features (i.e. abundance,
composition, and behaviour), and (iii) modality of pollen
Our work evidences how the ecological heterogeneity
found at forest edges can generate small-scale patterns
of relative insect abundance that are the premise for local
variations in PBIs and plant fitness. Shifts in both
mutualistic and antagonist insects can influence PBIs across
such habitat gradients, contributing to generate
quantitative and qualitative patterns of plant fitness. Such
patterns reveal that in edge habitats plants can rely on a very
small ecological space to optimize their reproduction.
Therefore, the conservation of edge-dependent species
requires the preservation of the habitat complexity typical
of such transition contexts. Further work is needed to
evaluate the effect on PBIs of temporal habitat dynamics
involving these peculiar ecological contexts.
The following additional information is available in the
online version of this article—
Table S1. List and frequency of insects visiting flowers
of D. balbisii during the observation trials, and their
putative role in the context of the biotic interactions involving
the study plant.
Sources of Funding
D.G. was supported by Ministero dell’Istruzione,
dell’Universita e della Ricerca (funding source ex MURST
Contributions by the Authors
D.G. originally formulated the idea, and developed
methodology; D.G. and L.B. conducted fieldwork, D.G. and G.F.
performed statistical analyses; D.G., G.F. and L.B. wrote
Conflict of Interest Statement
The authors are grateful to Prof. Pietro Brandmayr
(Universita della Calabria) for help in identifying insects.
We also thank Antonella Bonacci, Giovanni Borda,
Angela Salzano, Marco De Simone, Luigi Spina, Vittoria
Tropea and Maurizio Vena for support in the field, and
Antonio de Giuseppe for help in cultivating plants.
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