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R E S E A R C H A R T I C L E Open Access
Effects of population structure on pollen flow,
clonality rates and reproductive success in
fragmented Serapias lingua populations
Giuseppe Pellegrino
*
, Francesca Bellusci and Anna Maria Palermo
Abstract
Background: Fragmentation of habitats by roads, railroads, fields, buildings and other human activities
can affect population size, pollination success, sexual and asexual reproduction specially in plants showing
pollinator limitation, such as Mediterranean orchids. In this study, we assessed pollen flow, selfing rates,
vegetative reproduction and female reproductive success and their correlations with habitat characters in nine
fragmented subpopulations of Serapias lingua.
To improve understanding of population structure effects on plant biology, we examined genetic differentiation
among populations, pollen flow, selfing rates and clonal reproduction using nuclear microsatellite markers.
Results: Smaller populations showed a significant heterozygote deficit occurred at all five nuclear microsatellite
loci, the coefficient of genetic differentiation among populations was 0.053 and pairwise F
ST
was significantly
correlated with the geographical distance between populations. Paternity analysis of seeds showed that most
pollen flow occurred within a population and there was a positive correlation between percentage of received
pollen and distance between populations.
The fruit production rate varied between 5.10 % and 20.30 % and increased with increasing population size,
while the percentage of viable seeds (78-85 %) did not differ significantly among populations. The extent of
clonality together with the clonal and sexual reproductive strategies varied greatly among the nine populations
and correlated with the habitats where they occur. The small, isolated populations tended to have high clonal
diversity and low fruit production, whereas the large populations with little disturbance were prone to have
reductions in clonal growth and increased sexual reproduction.
Conclusions: We found that clonality offers an advantage in small and isolated populations of S. lingua,where
clones may have a greater ability to persist than sexually reproducing individuals.
Background
Fragmentation of plant populations, the process by
which formerly continuous populations turn into
patches of different sizes, isolated from each other, may
have distinctive effects on populations: (1) affecting repro-
ductive success, (2) altering patterns of pollen-mediated
gene flow (pollen flow) and (3) affecting self-pollination
and vegetative propagation. Although many plant popu-
lations are naturally isolated and small, populations of
numerous plant species have become more isolated as a
result of the recent anthropogenic fragmentation of
habitats by roads, railroads, fields, buildings and other
human activities [1, 2].
Fragmentation and the abundance of a plant species
can have striking effects on the visitation rate and floral
constancy of its pollinators, with potentially major im-
pacts on the plant's reproductive success, reducing the
abundance and species richness of pollinators, altering
their foraging behaviour and limiting pollinator move-
ment among populations [3, 4]. Thus, plants receive
fewer flower visits suffering pollen limitation and re-
duction in reproductive success. Studies of local popu-
lation density and size clearly show that pollination and
reproductive success decrease in sparse and small pop-
ulations [5]. Reductions in reproductive success due to
* Correspondence: giuseppe.pellegrino@unical.it
Dept. of Biology, Ecology and Earth Sciences, University of Calabria, I-87036
Rende, (CS), Italy
© 2015 Pellegrino et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Pellegrino et al. BMC Plant Biology (2015) 15:222
DOI 10.1186/s12870-015-0600-8
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
reduced insect movements are particularly strong for
plants which show a high degree of dependence on
their pollinator mutualism (i.e. pollinator limitation) for
fruit production [6], such as Mediterranean deceptive
orchids [7].
Sexual reproduction is predominantly pollinator
dependent, even if it may sometimes be successfully guar-
anteed by asexual reproduction or self-pollination. Self-
pollinating populations are more likely to establish in
habitats where pollinators appear to be scarce, in which
population size is small [8], and in environments with lim-
ited opportunity for outcrossing [9].
The complex flower structures and pollination strategies
of orchids are the best-documented examples of selection
for outcrossing in flowering plants to avoid inbreeding.
However, auto-pollinating orchids are relatively frequent
in geographically isolated and/or pollinator-scarce envi-
ronments such as higher latitudes/elevations, coastal areas
and islands [10, 11], supporting the ‘reproductive assur-
ance’hypothesis in which selection favours increased
self-pollination to ensure the persistence of populations
in situations in which pollinator service strongly limits
reproduction [12]. Approximately 20 % of terrestrial
orchid species in which the pollination system has been
investigated are capable of auto-pollination [11, 13],
suggesting that autopollination is indeed common in
Orchidaceae [14].
In the plant kingdom reproduction can be assured by
vegetative reproduction, a typical asexual reproduction
whereby new individuals are formed without the produc-
tion of seeds, including the formation of new plants out of
rhizomes, bulbs or tubers. Vegetative propagation leads to
a clonal structure in which one clone (genet) may consist
of several individuals (ramets). The most obvious genetic
signature of vegetative propagation in a population is the
presence of repeated multilocus genotypes (MLGs) and, as
a consequence, heterozygosity and allelic diversity at each
locus are expected to increase [15]. Many orchid species
have the capacity for vegetative propagation which can
represent the prevalent pattern of population mainten-
ance. There are several patterns of vegetative reproduction
in orchids, varying between species possessing different
life forms [16]. The most widespread pattern of vegetative
multiplication in orchids is the formation and germination
of two or more buds, including dormant ones, on axial
organs such as rhizomes, creeping shoots and shoot tu-
bers [17]. The daughter shoots are connected with the
maternal ones for a long time. The daughter shoots in
orchids with shoot rhizomes or bulbotubers (Anacamp-
tis,Dactylorhiza,Orchis,Ophrys,Serapias,etc.)separ-
ate most rapidly, after 1–2 years [18]. Among orchids
we can distinguish those with obligate vegetative propa-
gation, those with facultative vegetative propagation,
which includes short-rhizome and most tuberoidous
orchids, and those with vegetative propagation occur-
ring in exceptional cases [16].
An explicit method to clarify and quantify the direc-
tion of pollen flow between populations and to verify
the presence of spontaneous self pollination or vegeta-
tive reproduction is the molecular analysis of plants
and paternity analysis of seeds collected from known
mothers to determine the origin of the pollen that fer-
tilized the ovules.
In this study, we assessed pollen flow, selfing rates,
vegetative reproduction and female reproductive suc-
cess in nine fragmented subpopulations of an orchid
species, Serapias lingua. This species dependent upon
insect pollinators to ensure its reproduction, is self-
compatible and able to vegetatively reproduce [19] and
thus, is suitable for investigating the effects of popula-
tion fragmentation on gene flow, selfing/clonality rates
and reproductive success.
More specifically, we aimed at (1) determining the gen-
etic population structure to quantify clonality rates; (2)
examining fruit production rates in the studied popula-
tions to obtain estimates of female reproductive success;
and (3) examining a paternity analysis of seeds collected
from the plants.
Methods
Study species
The genus Serapias L. is distributed throughout the
Mediterranean region with its centre of diversity in
southern Italy and on the Greek islands [20].
Serapias lingua (tongue orchid) is a short-lived tuber-
ous orchid and a tetraploid species [21]. It has dull-
coloured flowers of uniform structure: the all three sepals
and the hypochile (the proximal part of the lip) form a
hood (tubular corolla), a unique shiny, more or less round
callosity, is present at the base of the hypochile, the
epichile (the distal part of the lip) is generally inclined
downwards. The petals and lip are characterized by con-
ical epidermal papillae and two types of trichome with
secretory apical cells [22]. It is a widespread species,
mainly distributed in the Mediterranean-Atlantic coun-
tries (Portugal, Spain, France, Italy, Balkans, Greece), but
reaching western North Africa (Morocco, Tunisia). It
grows in arid or wet meadows, abandoned agricultural
soils, garigue and bushy environments up to 1200 m a.s.l.
[23]. Recent molecular analysis strongly supports a nat-
ural split of S. lingua into a subgroup strictly related to
S. gregaria and S. olbia, two rare endemics of the Var
and Maritime Alps regions [24].
In the last years the pollination strategy of S. lingua
has received more attention, and preliminary observa-
tions indicate that Ceratina cucurbitina males are the
main pollinators [14, 25]. While other Serapias species
offer insects a floral tube in which to rest or sleep (shelter
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imitation strategy), S. lingua seems to have evolved to
sexually deceive pollinators, analogous to what is observed
in Ophrys orchids [26], a phenomenon also supported by
the finding of large amounts of alkanes and alkenes in its
floral odour extracts [27, 28].
Study area and measures of population size and density
The research site is located in southern Italy (Calabria
region). It covers approximately 700 ha and consists of
calcareous, dry grasslands (Festuco-Brometalia); Spartium
junceum L., Cytisus sessilifolius L. and Cistus incanus L.
are frequent shrubs and Festuca circummediterranea
Patzke,BromuserectusHuds. and Dactylis glomerata L.
are the dominant herbs.
Serapias lingua grows over the entire area, forming
populations of a few to thousands of individuals. We
define ‘a population’here as a group of S. lingua individ-
uals in a discrete area, each of which is separated from a
neighbouring population by at least 300 m (Fig. 1). A
total of 9 populations were identified; three (C, F, G) are
found in a highly anthropic landscape context enclosed by
busy roads and their intersections, while the remaining six
(A, B, D, E, H, I) are non-anthropic (natural) populations.
No other population is present in or around the study area
and the nearest population outside the study area is about
5 km north of population A.
In Spring 2014 the population size (i.e. the total num-
ber of individuals in a specific area) and population
density (i.e. the population size divided by total area)
was determined for each population. For population
size, we individually marked and counted the number
of all (flowering and vegetative) individuals in the three
smaller populations (C, F, G), while within each other
populations we marked and counted the number of
individuals in five selected square grid (10 by 10 m size)
separated by 30–50 m. The measurements resulting
from the five plots for each population were grouped
and used to calculated population size. For population
density, we calculated the area of the population (in
square metres) identifying the boundaries of each popu-
lation using the outermost individuals (Table 1). Vou-
cher specimens were deposited at the herbarium at the
University of Calabria (CLU).
Measures of reproductive success
To test natural reproductive success, in the three smaller
populations and in five square grid for each of the
remaining six populations, the number of flowers that
produced fruits was counted and the fruit set was de-
termined as the average of ratios (number of produced
fruits/number of available flowers) over the examined
plants. To ascertain the presence of viable embryos, at
least 1000 seeds from each fruit were removed from the
centre of the capsule and observed under an optical
microscope (100x). Seeds were assigned to two categor-
ies (viable and unviable seeds) due to the presence or
absence of viable embryos. The seed set [(the number
of filled seeds in sampled fruits/the number of observed
seeds) × 100] were calculated for every fruit.
In addition, in each population five individuals with
unopened flowers were bagged with a fine-meshed cloth
to exclude pollinators to test for spontaneous autogamy.
In June, the number of produced fruits was counted, and
the ratio between the number of fruits/treated flowers
was determined.
Fig. 1 Spatial distribution of Serapias lingua populations. Red areas indicate the nine populations defined by this study. Arrows represent pollen
flow and the numbers by the arrows indicate the numbers of pollen migration events. Figure was created by G. Pellegrino (the first author)
Pellegrino et al. BMC Plant Biology (2015) 15:222 Page 3 of 10
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DNA extraction and microsatellite genotyping
One leaf from each individual in the three smaller popu-
lations and from each individual in the five selected
areas of other six populations was sampled and stored in
silica gel for subsequent DNA extraction and microsatel-
lite (Short Sequence Repeat, SSR) genotyping. Genomic
DNA was extracted using a slight modification of the
CTAB (cetyltrimethyl ammonium bromide) protocol of
Doyle and Doyle [29]. Approx. 0.5 g of each leaf were
separately pestled in a 2 ml-Eppendorf vial using 500 μL
of standard CTAB buffer, incubated at 60 °C for 30 min,
extracted twice by adding 500 μL chloroform-isoamyl
alcohol (24:1), precipitated with isopropanol and washed
with 250 μL of ethanol 70 %. The DNA was re-suspended
in 50 μL of distilled water.
To characterize the genetic structure of each population
and genotype, we performed microsatellite genotyping on
all the adult plants using five nuclear microsatellite loci
previously isolated and tested on Serapias sp. [19, 30]. All
PCR reactions of 100 μl final volume contained 40 ng of
genomic DNA, 100 μM of each dNTP, 0.3 μMofeach
primer, 2 units of Taq polymerase, 2 μM MgCl
2
and 10 μl
of reaction buffer. The amplification conditions were:
1 cycle of 94 °C for 3 min;30 cycles 30 s at 94 °C, 45 s at
the locus specific annealing temperature (55 or 58 °C),
and 30 s at 72 °C using a Perkin Elmer thermal cycler.
One of the PCR primers for each locus was labeled with
fluorescent dye (FAM, TET). Labelled PCR products were
run together with the internal size standard GeneScan
ROX400 on an ABI 3110 (Perkin Elmer, Biosystems), and
individuals were genotyped using Genescan Analysis soft-
ware and Genotyper software (Perkin Elmer, Biosystems).
Clonality rates
Multilocus genotypes (MLGs) were assigned manually.
Because individuals with the same MLG found in popu-
lations with both sexual and vegetative reproduction can
be either ramets of the same genet or derive by chance
from distinct events of sexual reproduction, we used the
program GIMLET 1.3.2 [31] to estimate the probability
that two individuals, randomly sampled from a popula-
tion, shared the same MLG by chance (probability of
identity: PI).
Two different genotypic diversity indexes were calcu-
lated. The first measure was G/N, the ratio between the
number of MLGs and the total number of individuals in a
population [32]. Values of this index vary from zero (strict
clonality) in which all individuals share the same MLG, to
one (sexual reproduction) in which each individual has a
distinct MLG. The second measure was MLG diversity
(D
G
) [33] which measures the probability that two individ-
uals randomly selected from a population of N individuals
will have different MLGs. Similar to the first measure, D
G
ranges from zero indicating that there is only one domin-
ant clone, to one suggesting that every individual has a
different genotype.
Genetic variability
Population genetic analyses were based on a ‘corrected’
dataset in which all individuals with the same MLG were
considered as ramets of a single genet. For nSSRs, the
number of alleles, number of alleles per locus (N
a
) and
per population (N
ap
) [34], observed heterozygosity (H
O
),
gene diversity (H
E
) [35], and fixation index (F
IS
=1 –
H
O
/H
E
) were calculated for each locus and each popula-
tion using FSTAT version 2.9.3.2 [36]. Departures from
Hardy–Weinberg equilibrium at each locus and linkage
disequilibrium between loci were tested by an exact test
using a Markov chain method implemented in GENE
POP version 4.0 [37], with Bonferroni corrections. H
T
and H
S
[35], and F
ST
[38] were estimated using FSTAT.
H
T
is the gene diversity in the total population, H
S
is the
average gene diversity within populations, and F
ST
is
the coefficient of genetic differentiation among popula-
tions under an infinite allele model. Pairwise F
ST
values
were tested for significance by permuting genotypes
among populations. To test for the presence of isolation
by distance, a Mantel test between population-pairwise
Table 1 Population size and density, fruit production rate, percentage of viable seeds, immigration rate by pollen per population
Population Pop area
(in square
meters)
Pop size Pop density Fruit set (%) Viable seeds (%) Immigration rate
by pollen (%)
Pollen source population
ABCDEFGHI
A 3578.25 ~2800 0.78 13.58 82.78 ± 3.73 28.68 553 6 114 112
B 2540.20 ~2000 0.79 20.30 79.85 ± 2.44 32.02 22 571 203 24
C 64.20 302 4.70 5.20 78.55 ± 2.13 9.38 1 29 1
D 3451.22 ~3000 0.87 14.23 81.21 ± 2.86 28.34 151 610 8 1 81
E 2962.40 ~2500 0.84 15.60 85.35 ± 3.83 27.49 102 8 15 565 84 6
F 55.80 321 5.75 5.50 81.21 ± 3.27 11.11 2 32 2
G 65.54 284 4.31 5.10 82.24 ± 2.33 7.14 2 26
H 4585.30 ~3200 0.70 14.68 82.54 ± 3.66 30.53 40 11 209 609 6
I 2542.60 ~2200 0.86 16.75 79.65 ± 2.05 28.64 12 182 9 1 535
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geographic distance and F
ST
/(1 –F
ST
) was applied [37].
Null allele (alleles that did not give a polymerase chain
reaction product) frequencies were estimated using the
maximum-likelihood (ML) estimator based on the EM
algorithm and implemented by default in GENEPOP 4.0
[37]. Based on microsatellite allele frequencies, recent
population bottlenecks were checked by BOTTLENECK
[39], employing the Two Phase Mutation model (TPM)
with a 95 % Stepwise Mutation Model (SMM) and 5 %
multistep mutations. Significance was assessed using
the Wilcoxon test. The bottleneck program [40] was
used as an alternative measure of genetic bottlenecks to
test for excess gene diversity relative to that expected
under mutation-drift equilibrium. The heterozygosity
excess method exploits the fact that allele diversity is
reduced faster than heterozygosity during a bottleneck,
because rare alleles are lost rapidly and have little effect
on heterozygosity, thus producing a transient excess in
heterozygosity relative to that expected in a population
of constant size with the same number of alleles [39].
Paternity assignment
Microsatellite profiles for each fruit were also determined
to ascertain if fruit developed by plants in each population
could have been produced by pollen transferred by
individuals of the same population or different donors.
In June, capsules were collected and seeds in the cen-
tral part were used for molecular analysis. Seeds were
observed under a binocular microscope and approx. 50
viable seeds (which means seeds with an embryo) from
each capsule were collected and transferred into single
2 ml-Eppendorfs to extract their DNA. Nuclear microsat-
ellite loci were amplified and analyzed following the proto-
col described above. Paternity analysis was performed by a
likelihood-based approach based on multilocus genotypes
for all adult genets and offspring using CERVUS version
2.0 [41]. In this study, the simulation parameters required
by the program were set as follows: 10 000 cycles, 4956
candidate parents (= all fruits collected across the study
population), 0.99 as the proportion of candidate parents
sampled, and 1.00 and 0.001 as the proportions of loci
typed and mistyped, respectively.
According to the assigned paternity data, we catego-
rized the fruit as derived from selfing, outcrossing within
the study area, and outcrossing with a paternal parent
that was not present in the study area. We defined the
selfing rate as the number of selfed fruits divided by the
number of examined fruits from each population.
Results
Population size and density
The stands differed in population size, ranging from
284 to ~3200 individuals, in population density (0.70–5.75
individuals/m
2
) (Table 1) and degree of isolation (the
distance between S. lingua populations ranged from
300 m to 2.5 km). Three populations (C, F, G) showed
significantly lower values of population size and higher
values of population density than the other six popula-
tions, such as they had lower population areas (Table 1).
Reproductive success
Significant differences were detected among the popula-
tions in their fruit production rate. Indeed, the popula-
tions differed significantly in their fruit sets, which
varied from 5.10 % to 20.30 % and was 14.53 % for the
nine populations on average. More specifically, the three
smallest populations in term of population size (C, F, G)
showed lower values than the other populations, which
showed values four times higher (Table 1). In contrast,
the populations did not differ significantly in their per-
centage of viable seeds, which varied from 78.55 (±2.13)
for population C to 85.35 (±3.83) for population E
(Table 1). The best explanation for the variation in the
fruit production rate is the positive correlation between
fruit set and population size. Indeed, the estimated par-
ameter for the population size was positive, suggesting
that larger populations have higher outcrossing rates.
None of the 45 individuals (five per population) bagged
with a fine-meshed cloth to exclude pollinators showed
any spontaneous autogamy.
Presence and extent of clonal propagation
All populations were affected by different levels of
clonality. The population with the lowest G/N ratio
was C (0.067), and slightly higher values were shown
by the other two (F and G) small populations
(Table 2). Higher G/N values were found in the other
populations, ranging from0.812(populationA)to
0.892 (population H). Similar results were found for
Table 2 Measures of clonal propagation: ratio between the
number of multilocus genotypes and the total number of
individuals (G/N), and multilocus genotype diversity (D
G
)in
nine populations of S. lingua
Population G/N D
G
A 0.812 0.721
B 0.885 0.748
C 0.067 0.038
D 0.862 0.740
E 0.854 0.725
F 0.085 0.040
G 0.088 0.041
H 0.892 0.794
I 0.886 0.784
mean 0.603 0.515
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multilocus genotype diversity (D
G
), which ranged from
close to zero (population C) to 0.794 (population H),
with a mean value of 0.215 (Table 2).
Genetic diversity and differentiation among populations
PCR products were successfully obtained from all ex-
amined individuals, their fragment lengths fit into the
predicted size ranges, and all examined loci were poly-
morphic across the nine populations. No significant link-
age disequilibrium between loci was observed for any
population, so all loci were used for further analyses.
The total number of alleles per population ranged
between 4 and 15 (average 9.6 alleles) and the number
of alleles per locus ranged between 8 and 20 (data not
shown). Three populations (C, F, G) had a lower mean
allele number per population than the other popula-
tions, and possessed all alleles exhibited by natural
populations. Moreover, in anthropic populations the
observed heterozygosity was much less than expected
(H
O
= 0.38-0.42;H
E
= 0.52-0.60), while the other popula-
tions possessed higher heterozygosity (H
O
ranging from
0.77 to 0.80) that was close to expected values (H
E
ranging from 0.75 to 0.79) (Table 3). Inbreeding
coefficients (F
IS
) calculated at each nSSR locus in each
population (45 values) varied among populations. Six
populations showed a low heterozygote excess ranging
from F
IS
=−0.02 (pop E) to F
IS
=−0.12 (pop A), while
three others showed a significant heterozygote deficit
(F
IS
= 0.22-0.28) at all five loci (Table 3). Few private
alleles were found in each population. The coefficient
of genetic differentiation among populations (F
ST
)was
estimated to be 0.053 for nSSRs. Pairwise F
ST
/(1 –F
ST
)
was significantly correlated with the geographical dis-
tance between populations for nSSRs (P < 0.05, Fig. 2).
Bottleneck analysis revealed that three populations
had a significantly higher observed gene diversity than
expected under the 95 % Stepwise Mutation Model,
while no deviation from mutation-drift equilibrium was
found for any other population. In a population at
mutation-drift equilibrium (i.e., the effective size has
remained constant in the recent past), there is an
approximately equal probability that a locus shows
either a gene diversity excess or a gene diversity deficit.
Populations that have experienced a recent reduction in
their effective population size exhibit a correlative reduc-
tion in the number of alleles and gene diversity at poly-
morphic loci. But the number of alleles is reduced faster
than the gene diversity. Thus, in a recently bottlenecked
population, the observed gene diversity is higher than the
expected equilibrium gene diversity computed from the
observed number of alleles, under the assumption of a
constant-size (equilibrium) population [42].
Paternity assignment of seeds
In the paternity assignment experiments, 4967 fruits were
obtained from 5176 plants in nine populations (Table 1).
DNA extraction failed for 21 samples, but the paternity of
the remaining 4956 was examined and identified at a 95 %
confidence level. There was significant differentiation by
the paternity test among populations in term of the per-
centage of immigration rate, which varied from 7.14 %
(population G) to 32.02 % (population B). Indeed, in six
populations (A, B, D, E, H and I) the pollen parents of
approx 30 % of the fruit were located outside each
population, and the remaining 70 % within the popula-
tion,whileinthreepopulations(C,F,G)thepollenpar-
ents of ~90 % and ~10 % of the fruit were located
within and outside each population, respectively. The
mother plants of populations A, B, D, E, H and I received
pollen widely from other populations. The maximum
pollen dispersal distance within the whole population was
1100 m. Interestingly, there was a positive correlation
between the percentage of received pollen and the dis-
tance between populations (Fig. 1). Indeed, greater gene
flow occurred between the nearest populations, while gene
flow was close to zero among the most distant popula-
tions. No fruits were produced by selfing.
Discussion
Population genetic structure
In this study analysis of microsatellite DNA variation in
Serapias revealed clear and significant genetic differen-
tiation among populations, suggesting different levels
of gene flow between them.
In our investigations the number of alleles per locus
(8–18) and the mean of 9.6 alleles per population are
higher values than the alleles per locus (4–10) and
alleles per population (3.6-5.6) detected by Pellegrino et
al. [19, 43] in populations of other Serapias species (S.
parviflora,S. politisii and S. vomeracea). But these values
are similar to or slightly lower than those reported to date
Table 3 Measures of number of alleles per population (N
ap
),
observed (H
O
) and exptected (H
E
) heterozygosity, and fixation
index F
IS
in nine populations of S. lingua
Population N
ap
H
O
H
E
F
IS
A 15 0.784 0.774 −0.12
B 9 0.774 0.752 −0.04
C 4 0.418 0.594 0.25
D 10 0.789 0.755 −0.07
E 11 0.776 0.762 −0.02
F 6 0.422 0.524 0.28
G 5 0.382 0.516 0.22
H 14 0.782 0.789 −0.08
I 12 0.777 0.778 −0.04
Average 9.6 0.656 0.694 0.04
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for other Mediterranean orchid genera, Dactylorhiza
[44], Gymnadenia [45, 46], and Ophrys [47, 48].
The five markers included in this study showed
medium levels of genetic variation (H
E
ranging from
0.69 to 0.79, average 0.694) compared with other micro-
satellite studies on orchids [47].
The low value of genetic differentiation among popula-
tions (F
ST
=0.053) is due to the small geographic range of
the S. lingua populations studied. Indeed, similar genetic
differentiation values based on microsatellites have been
reported in other small orchid populations of Caladenia
huegelii [49] and Gastrodia elata [50], showing geographic
distances of 150 and 250 km, respectively.
Patterns of population genetic diversity and viability
may vary greatly across populations due to a multitude
of possible variables [51]. Populations may lose most of
their genetic diversity if they become very small and iso-
lated [52]. Accordingly, we detected two distinct groups;
first group formed by the three smallest S. lingua popu-
lations (C, F, G) showed a substantial deficit in genetic
diversity, the largest difference between observed and
expected heterozygosity, and higher values of inbreed-
ing coefficients (F
IS
), while the second group formed by
the other populations possessed observed heterozygos-
ity close to expected heterozygosity values and lower
values of inbreeding coefficients (Table 3). The genetic
poorness of smaller populations often derives from lim-
ited connections to other populations [53].
Paternity test and gene flow
Data from the paternity test of seeds showed that there
were high frequencies of short-distance and low frequen-
cies of long-distance pollen dispersal events. In the study
populations, greater gene flow occurred between the
nearest populations (distance from 300 to 500 m), while
the rate of gene flow decreased in populations farther
from each other (distance from 1000 to 1500 m) and
there was little or no inter-population gene flow between
the three smallest and most isolated populations (Fig. 2).
In addition, these three populations showed that the
flowers were pollinated in 90 % of cases by the pollen of
the same population and only 10 % by pollen from other
populations, which in contrast showed a greater flow of
pollen input. Pollination events between populations
increased with the geographical separation of the popula-
tions, suggesting that most movements of pollinators
occur within populations. This is probably a consequence
of inadequate pollinator visitation to small populations,
resulting in strong gene flow limitation [2, 54]. The greater
flow of pollen between the nearest populations is in agree-
ment with the behaviour of pollinators. Indeed, recent
work based on the capture and recapture of pollinating
insects showed that the average distance travelled by polli-
nators was 300 m, and only a few insects were recaptured
at distances of approximately 1000 m [55]. But this does
not explain the lower pollen flow from outside the smaller
populations in comparison with the larger populations,
independent of the distance between the populations.
Probably, there are other factors that determine this
reduction. For example, one factor may be the popula-
tion size, since the examined populations showed that
proportions of out-of-plot pollen flow were positively
correlated with the number of adult plants within the
population. Larger populations of plants are likely to be
more attractive to pollinators, resulting in higher visit-
ation rates, whereas small fragmented populations may
be less attractive [56]. In addition, a population with a
longer perimeter will likely have more insects (i.e. polli-
nators) encounter it, resulting in increased pollination.
Moreover, a higher population density can result in
greater pollination between individuals in the same
population or an increase in the selfing rate [57]. In our
Fig. 2 The correlation between pairwise F
ST
/(1 –F
ST
) and geographical distance
Pellegrino et al. BMC Plant Biology (2015) 15:222 Page 7 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
case, as the species is self-compatible, but not capable
of producing fruits via spontaneous autogamy, the de-
tected patterns can only be the result of active pollen
transfer by pollinators, and thus the pollination success
of S. lingua was significantly and positively related to
population size. This is in accordance with the outcome
of several studies on orchids that have already shown
that gene flow is often positively affected by increasing
population size [58]. In addition to the population size,
our study indicated that the population density of flower-
ing plants also affected pollinia removal, which increased
when the local density decreased. This data is in apparent
contrast with many previous papers on food-deceptive or-
chids, and in agreement with studies on sexually deceptive
orchids. Indeed, Vandewoestijne et al. [59] showed that
pollinator activity generally increased with decreasing
population density in three Ophrys species, suggesting that
pollinator availability, rather than pollinator learning, is
the most limiting factor in successful pollination for sexu-
ally deceptive orchids. Moreover, in sexually deceptive or-
chids, insects rarely switch from one individual to another
close individual immediately after the first attempted
copulation, preferring to fly off at a greater distance from
the first individual [60], suggesting that the apparent
avoidance of multiple copulations within a small popula-
tion will promote pollen flow over a greater distance [61].
Sexual reproductive success and clonality rates
The results reported here showed that clonality repre-
sents a common reproductive strategy in all analysed
populations, but clonality did not affect the different
populations of S. lingua equally. Six larger S. lingua popu-
lations showed higher levels of clonality (DG = 0.71-0.79),
for example, similar to those found in the endangered spe-
cies Cypripedium calceolus (DG = 0.97; [62]), while the
lowest clonal diversity (G/N index) and reduced heterozy-
gosity (H
O
= 0.38-0.42) in smaller populations, similar to
those found in polish Epipactis atrorubens [63] and
Cephalantera rubra populations [64], was a consequence
of particularly intensive vegetative reproduction. Ac-
cording to our data, the C, F, and G populations
showed a higher rate of clonality, while in other popu-
lations sexual strategies seemed to contribute more to
reproduction. A hypothesis that may explain the pat-
tern of clonality that we found in smaller populations is
low sexual reproduction in these populations due to
pollinator limitation, as evidenced by the small number
of fruits produced. The balance between sex and clonal
growth varies between and within species and is mainly
driven by biotic and environmental factors [65]. Although
vegetative propagation has ecological costs related to
greater resource uptake, reduced pollen dispersal, or in-
creased geitonogamous pollination [66], species showing
higher rates of clonality have several potential ecological
and evolutionary advantages. In our case, S. lingua can
persist in small, isolated populations where conditions
are not favourable for sexual reproduction, providing a
form of reproductive assurance by guaranteeing the
survival of the species in case of limited pollinator
service [15]. Thus, the combination of the availability of
pollinators and the fruit set related to population size
characterizing each population and the distance between
neighbouring populations of S. lingua can explain the
different levels of clonal propagation we found in differ-
ent populations. In particular, a higher rate of asexual
reproduction was found in C, F, and G than in other
populations, the former consisting of a few hundred
individuals located in a restricted area (about 70 m
2
)
closed to a crossroads, the latter comprising a thousand
individuals in a larger area (~0.5 ha). Populations sub-
jected to more environmental stress and fragmentation
by roads, railroads, fields, buildings and other human
activities show higher levels of clonality [15, 67].
Conclusions
This study represents one of the few analyses of the effects
of population structure on the pollen flow and clonal
growth of a deceptive Mediterranean orchid. Population
fragmentation is likely to reduce reproductive success due
to reductions in population sizes and increases in the
geographic distance between populations. We found that
clonality offers an advantage in small and isolated pop-
ulations of S. lingua, whereby clones may have a greater
ability to persist than sexually reproducing individuals
[61]. Since clonal growth is associated with a progressive
reduction in genotypic diversity, sexual reproduction
might be indispensable to the long-term success of a
species and clonal growth may play an important role
in prolonging the time to extinction when sex is
reduced or absent.
Abbreviations
CTAB: Cetyltrimethyl ammonium bromide; D
G
: Multilocus genotype diversity;
F
IS
:Fixationindex;F
ST
: Coefficient of genetic differentiation among populations;
H
E
:Genediversity;H
O
: Observed heterozygosity; H
S
: Average gene diversity
within populations; H
T
: Gene diversity in the total population; ML: Maximum-
likelihood; MLG: Multilocus genotypes; N
a
: Number of alleles per locus;
N
ap
: Number of alleles per population; PI: Probability of identity; SMM: Stepwise
mutation model; SSR: Short sequence repeat; TPM: Two phase mutation.
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
GP conceived of the study, and participated in its design and coordination
and was the key person writing the manuscript. FB carried out the
molecular genetic studies. AMP performed the statistical analysis and
participated in writing of the manuscript. All authors read and approved
the final manuscript.
Authors’information
All authors belong to the Department of Biology, Ecology and Earth
Sciences, University of Calabria, I-87036 Rende (CS), Italy
Pellegrino et al. BMC Plant Biology (2015) 15:222 Page 8 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Acknowledgements
This work was supported by grants to GP and AMP from the University of
Calabria, Department of Biology, Ecology and Earth Sciences.
Received: 27 April 2015 Accepted: 2 September 2015
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