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Plant Ecology & Diversity
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Population genetics and conservation of the
extremely narrow Pyrenean palaeoendemic Glandora
oleifolia (Boraginaceae)
Alberto del Hoyo a , Jordi López-Pujol b c , Mi Yoon Chung d & Blanca Lasso de la Vega e
a Marimurtra Botanic Garden, Blanes, Spain
b BioC-GReB, Laboratory of Botany, Faculty of Pharmacy, University of Barcelona,
Barcelona, Spain
c BioC-GReB, Botanic Institute of Barcelona (IBB-CSIC-ICUB), Barcelona, Spain
d Department of Biology and Research Institute of Natural Science, Gyeongsang National
University, Jinju, Republic of Korea
e Jardín Botánico-Histórico La Concepción, Málaga, Spain
Accepted author version posted online: 30 Oct 2012.Version of record first published: 05
Nov 2012.
To cite this article: Alberto del Hoyo , Jordi López-Pujol , Mi Yoon Chung & Blanca Lasso de la Vega (2012): Population
genetics and conservation of the extremely narrow Pyrenean palaeoendemic Glandora oleifolia (Boraginaceae), Plant
Ecology & Diversity, 5:4, 501-511
To link to this article: http://dx.doi.org/10.1080/17550874.2012.735270
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Plant Ecology & Diversity
Vol. 5, No. 4, December 2012, 501–511
Population genetics and conservation of the extremely narrow Pyrenean palaeoendemic Glandora
oleifolia (Boraginaceae)
Alberto del Hoyoa$, Jordi López-Pujolb,c*$, Mi Yoon Chungd, and Blanca Lasso de la Vegae
aMarimurtra Botanic Garden, Blanes, Spain; bBioC-GReB, Laboratory of Botany, Faculty of Pharmacy, University of Barcelona,
Barcelona, Spain; cBioC-GReB, Botanic Institute of Barcelona (IBB-CSIC-ICUB), Barcelona, Spain; dDepartment of Biology and
Research Institute of Natural Science, Gyeongsang National University, Jinju, Republic of Korea; eJardín Botánico-Histórico La
Concepción, Málaga, Spain
(Received 13 December 2011; final version received 26 September 2012)
Background: Glandora oleifolia is one of the most narrowly restricted endemics of the Iberian Peninsula. It occurs in two
locations, separated by 5 km in the south-eastern Pyrenees. The species is listed as ‘vulnerable’ in the Spanish Red List, and
it is legally protected in Catalonia.
Aims: To study the genetic diversity and population structure, and to review the conservation status and the threats to the
survival of this species.
Methods: The genetic variation of the two extant subpopulations was surveyed by means of allozymes and random amplifi-
cation of polymorphic DNA (RAPD).
Results: Both markers indicated depleted levels of genetic variability for G. oleifolia, in agreement with the expectation for
endemic species, and low genetic divergence among the two subpopulations (below 10% with the two nuclear markers).
Conclusions: The evolutionary history of this palaeoendemic species – repeated population bottlenecks and/or local
extinction of populations during the glacial periods of the Quaternary – could have enhanced the loss of its genetic diversity.
Keywords: allozymes; extinction; genetic structure; narrow endemic; RAPD; rare species
Introduction
The Mediterranean Basin is regarded as one of the most
important hotspots of global biodiversity because of its
enormous floristic richness (it harbours ca. 10% of the
total number of the higher plants of the world in an area
encompassing just 1.6% of the Earth’s surface) and excep-
tional rates of endemism, with 13,000 of the 25,000 plant
species occurring in the Basin being endemics (Médail and
Quézel 1997; Myers et al. 2000). A major fraction of the
Mediterranean endemic species (nearly 60%) are narrow
endemics, that is, taxa that are often restricted to an island
or a mountain range (Thompson 2005). This wealth of local
endemism has been attributed to the heterogeneous evo-
lutionary history of Mediterranean plant lineages, which
has resulted from the interaction between the most recent
geological and climatic events of the Basin (e.g. Alpine
orogeny, microplate split-off and dispersal, the Messinian
salinity crisis, or Pleistocene climatic oscillations) and its
large geographical heterogeneity (the myriad of islands and
mountain ranges occurring along the Mediterranean consti-
tute strong barriers to plant dispersal) (Verlaque et al. 1997;
Blondel and Aronson 1999; Thompson 1999, 2005; Médail
and Diadema 2009).
Because Mediterranean narrow endemics tend to grow
in sites with high rock cover and pronounced slopes, such
*Corresponding author. Email: jlopezpu@gmail.com
$These authors equally contributed to this work.
as cliffs (Lavergne et al. 2004; Thompson 2005), it is not
surprising that mountain ranges (both mainland and insu-
lar) represent important areas of plant endemism within the
Basin (Favarger 1972; Médail and Quézel 1997; Blondel
and Aronson 1999). The Pyrenees, in addition to harbour-
ing high rates of endemism (over 10%; Favarger 1972;
Médail and Quézel 1997), also constituted one of the
main glacial refugia for Mediterranean plants (Dubreuil
et al. 2008; Médail and Diadema 2009). Rocky cliffs and
deep gorges are very common along the Pyrenees and
adjacent mountain ranges (such as the Pre-Pyrenean belt
and the Catalanidic Mountains) and constituted relatively
stable habitats during the extreme Quaternary climatic
fluctuations because of their sheltered environmental con-
ditions, providing for example continued moisture avail-
ability (Thompson 2005; Médail and Diadema 2009). Many
Tertiary lineages that today are some of the most conspic-
uous examples of narrow endemism in the Pyrenees and
in neighbouring mountains, such as Borderea chouardii
(Gaussen) Heslot (Dioscoreaceae) or Ramonda myconi (L.)
Rchb. (Gesneriaceae), could have escaped from extinction
by surviving in these rocky habitats, either in unchanged
form or by differentiating into new species (Martínez-
Rica and Montserrat-Recoder 1990; Dubreuil et al. 2008;
Segarra-Moragues and Catalán 2008).
ISSN 1755-0874 print/ISSN 1755-1668 online
© 2012 Botanical Society of Scotland and Taylor & Francis
http://dx.doi.org/10.1080/17550874.2012.735270
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502 A. del Hoyo et al.
Glandora oleifolia (Lapeyr.) D.C. Thomas
[≡Lithodora oleifolia (Lapeyr.) Griseb.] is one of the
narrowest rocky endemics of the Pyrenean mountains and
surely one of the rarest species of Boraginaceae in the
world. Its occurrence is restricted to just two subpopula-
tions (separated by less than 5 km) in the eastern Pyrenees
(Catalonia, north-eastern Spain; Figure 1), growing on
limestone and totalling less than 10,000 individuals. The
species is included as ‘vulnerable’ in both the 2008 Spanish
Red List (Moreno 2008) and the Catalonia Red Book (Sáez
et al. 2010), and it is strictly protected in Catalonia (DOGC
2008). Glandora is a small genus recently segregated
from Lithodora (Thomas et al. 2008), and comprises just
six species, all distributed in the Western Mediterranean
Basin. The genus diverged from its sister group ca. 20 Ma,
and speciation within Glandora could have taken place
during the middle and late Miocene (Weigend et al. 2009).
G. oleifolia is the basal species of the genus (Ferrero et al.
2009).
To survey the genetic diversity of rare, endemic or
threatened plant species, the combination of several molec-
ular markers (instead of a single marker) is highly rec-
ommended. This approach provides a larger number of
loci from different regions of the genome. Allozymes
and random amplification of polymorphic DNA (RAPD)
are among the most-used types of markers in population
genetic studies because of their relative technical simplic-
ity and low cost (Lowe et al. 2004; Coates and Byrne
2005). The codominant inheritance of allozymes allows the
inference of genetic variation at genetic loci with reliabil-
ity, although they have the drawback that they represent a
highly biased part of the genome (just a few dozen loci
encoding well-documented, soluble proteins; Aagaard et al.
1998; Lee et al. 2002). RAPDs, in contrast, are dominant
markers, an inheritance mode that may produce biases in
some population genetic parameters (Aagaard et al. 1998;
Torres et al. 2003). Another problem derived from the use
of RAPD markers is the reliability of fingerprint patterns.
Penner et al. (1993) found that the main factor regarding
the reliability of RAPD analyses are not the samples, but
the laboratory reagents and equipment, the thermal cycler
being the main source of variation. Nevertheless, RAPDs
can yield a very large number of loci that are supposed to
provide a much more random sample of genomic DNA than
allozymes (Lynch and Milligan 1994); moreover, since they
are the products of amplification of primarily non-coding
DNA regions, they are expected to be more susceptible to
mutations and less sensitive to selection than allozyme loci
(Ayres and Ryan 1997; Aagaard et al. 1998; Nybom and
Bartish 2000).
Therefore in combination, these two types of mark-
ers can provide a broad picture of the genetic diversity
of targeted plant species, such as G. oleifolia, because
the two types exhibit complementary properties. In this
study, allozymes and RAPDs were employed to estimate
genetic variation and genetic structure of this palaeoen-
demic species. Given its rarity, we can predict low lev-
els of genetic diversity for this species; alternatively, one
may expect high levels of genetic variation as the species
grows in a region that harboured plant refugia during the
Pleistocene. A further aim of this study was to review the
current threats and conservation status of the species.
Figure 1. Extant and extinct subpopulations of Glandora oleifolia. The black stars denote the extant ones, which were sampled for the
present study, whereas the grey stars indicate those that disappeared during the twentieth century. SAN, Sant Aniol d’Aguja (Catalonia,
Spain); TOL, Toll de Monars (Catalonia, Spain); BEG, Beget (Catalonia, Spain); LAM, Lamanère (Eastern Pyrenees Dept., France).
Glaciated areas during the Last Glacial Maximum (LGM) are dotted, according to Calvet (2004). The map is reproduced with permission.
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Population genetics and conservation of Glandora oleifolia 503
Materials and methods
Plant material and sampling strategy
Glandora oleifolia is a small evergreen shrub with decum-
bent stems that grows in fissures of limestone rocks at
altitudes of 400–900 m in the Alta Garrotxa region in
the eastern Pyrenees, Catalonia, north-eastern Spain. Its
diploid chromosome number is 2n =28 (Luque and Valdés
1984). Flowers are actinomorphic and pentamerous, with a
blue corolla 14–20 mm in length, and they exhibit distyly:
both long-styled morphs (flowers with long styles and short
stamens) and short-styled morphs (flowers with short styles
and long stamens) occur. Pollination of this species is ento-
mophilous, the main pollinators being the long-tongued
Antophora (Hymenoptera) and Bombylius (Diptera). The
species is self-compatible (Ferrero 2006; Ferrero et al.
2011) and, in fact, it has a mixed-mating system (Ferrero
et al., 2012); it flowers in May and June.
Samples were collected from the two extant subpopu-
lations of G. oleifolia: Sant Aniol d’Aguja (SAN) and Toll
de Monars (TOL) (Figure 1). Young leaves were collected
on a linear transect within each subpopulation. Widely
separated individuals were chosen for sampling to avoid
collecting ramets from the same genet, since the individu-
als may produce large stolons. In all, 62 individuals from
SAN and eight from TOL were sampled for allozymes.
To sample for RAPD, we collected 22 individuals from
the SAN subpopulation and used the same eight individ-
uals (the only accessible ones) from TOL. Samples were
placed in envelopes, transported immediately to the lab-
oratory, and either stored at 4 ◦C until extraction 1 day
later (for allozymes) or dried and stored with silica gel
in zip-lock plastic bags until DNA extraction. Because of
the threatened status of this species, leaf samples were col-
lected carefully in order to minimise the potential damage
to the population.
Allozyme electrophoresis
Standard methods for starch gel electrophoresis of
allozymes (Soltis et al. 1983; Soltis and Soltis 1989)
were used to assess the genetic variability in Glandora
oleifolia. Leaves were homogenised on refrigerated porce-
lain plates using a cold extraction buffer, consisting of
0.05 M tris-citric acid, 0.1% (m/v) cysteine·HCl, 0.1%
(m/v) ascorbic acid, 1% (m/v) polyethylene glycol, 8%
(m/v) PVP-40 (polyvinyl-pyrrolidone), and 0.06% (v/v)
2-mercaptoethanol. Extracts were absorbed onto 3 MM
Whatman filter paper. They were either analysed imme-
diately or stored at -80 ◦C for long-term conservation
until electrophoresis. By using 11% starch gels, nine
enzyme systems were resolved and 13 interpretable loci
were obtained: Aat-1,Gdh-1,Gdh-2,Idh-1,Idh-2,Ldh-1,
Mdh-1,Mdh-2,Me-1,Pgi-2,Pgm-1,Pgm-2, and Skd-1.
Aspartate aminotransferase (AAT, EC 2.6.1.1), glutamate
dehydrogenase (GDH, EC 1.2.1.2), lactate dehydroge-
nase (LDH, EC 1.1.1.27), and malic enzyme (ME, EC
1.1.1.40) were satisfactorily resolved on pH 8.2 tris-citrate
buffer; phosphoglucoisomerase (PGI, EC 5.3.1.9) and
phosphoglucomutase (PGM, EC 5.4.2.2) were resolved
on pH 7.0 histidine buffer; finally, isocitrate dehydroge-
nase (IDH, EC 1.1.1.42), malate dehydrogenase (MDH,
EC 1.1.1.37), and shikimate dehydrogenase (SKD, EC
1.1.1.25) were resolved on pH 6.1 morpholine-citrate
buffer. Alcohol dehydrogenase (ADH, EC 1.1.1.1) showed
activity but was not scorable owing to poor resolution of
bands. Staining procedures for all enzymes followed the
method described by Wendel and Weeden (1989), with
slight modifications.
RAPD
Twenty RAPD primers (C, J, L, kits from Operon
Technologies) were assayed in a pilot sample with sev-
eral individuals from each of the two subpopulations.
Amplifications were carried out in 10 µl total volume con-
taining 1×PCR buffer (Bioline), 3.5 mM MgCl2(Bioline),
0.2 mM of each dNTP (Bioline), 0.2 µM of primer, 0.9 unit
of Taq DNA polymerase (Bioline), and 25 ng of template
DNA. The amplification programme consisted of a step of
DNA melting of 1 min at 92 ◦C, followed by 45 cycles
of 92 ◦C for 10 s, 35 ◦C for 10 s, and 72 ◦Cfor1min,
and a final elongation step of 72 ◦C for 2 min. Reaction
products were separated by electrophoresis in 2% agarose
gels stained with ethidium bromide; electrophoresis was
set at 100 V during 3 h in 0.25×NEB buffer. RAPD
bands were visualised with UV transmitted light, captured
with Kodak DC 120, and edited with Kodak DS 1D soft-
ware. To check the reproducibility of the banding profiles,
RAPD amplifications were repeated at least twice, follow-
ing the method proposed by Rieseberg (1996). Ten of the
20 assayed primers were selected based on band reliability,
clarity, signal strength, and resolution for the analysis of
the whole set of samples. All the analyses were performed
in the same laboratory and with the same Perkin-Elmer
thermal cycler to avoid reliability problems (Penner et al.
1993).
Genetic analysis of allozyme data
Beginning from the anode, loci were numbered consecu-
tively, and alleles at each locus were labelled alphabetically.
Interpretation of banding patterns was made on the basis of
the quaternary structure of isozymes, subcellular localisa-
tion and number of loci usually expressed in diploid plants
(Gottlieb 1982; Soltis and Soltis 1989). Allele frequencies
at each locus were calculated for each subpopulation. To
estimate the levels of genetic diversity, the following statis-
tics were calculated: P, the percentage of polymorphic loci
when the most common allele had a frequency of <0.95; A,
the mean number of alleles per locus; Ap, the mean number
of alleles per polymorphic locus; AR, the mean allelic rich-
ness using a ‘rarefaction method’ that compensates uneven
population sample sizes (El Mousadik and Petit 1996), Ho,
the observed heterozygosity; and He, the expected pan-
mictic heterozygosity. The mean fixation index (F) for all
polymorphic loci in each subpopulation was also calculated
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504 A. del Hoyo et al.
to compare genotype proportions with those expected under
Hardy–Weinberg equilibrium. The chi-square test (χ2)was
used to evaluate deviations of Ffrom zero. To measure
inbreeding within populations and genetic differentiation
among them, Wright’s (1965) F-statistics were computed:
the inbreeding coefficient (FIS), the fixation index (FST),
and the overall inbreeding coefficient (FIT). All these cal-
culations were carried out with POPGENE v1.32 (Yeh et al.
1997) and FSTAT v2.9.3.2 (Goudet 2002).
The genetic structure of the species was studied by
using an analysis of molecular variance (AMOVA) with
the software ARLEQUIN v3.1 (Excoffier et al., 2006), to
calculate variance components and their significance levels
(by permutation tests, using 1000 replicates) for varia-
tion within and among the two subpopulations. Genetic
structure was also assessed by means of the Bayesian algo-
rithm implemented in STRUCTURE 2.3.1 (Hubisz et al.
2009), which estimates the likelihood of the individuals
being structured in a given number of genetic clusters (or
genetic populations, K). This can be done in the absence of
previous population information (i.e. without pre-defined
populations), and the software probabilistically assigns
individuals to the genetic clusters. This method, contrary
to AMOVA, assumes that loci are at Hardy–Weinberg equi-
librium and linkage equilibrium. The programme was run
from K=1toK=5, and the admixture ancestry model
with correlated allele frequencies was selected as an appro-
priate option for the analysis. The burn-in period and
Markov Chain Monte Carlo (MCMC) were set to 50,000
and 100,000 iterations, respectively, and 10 replicates per
Kwere run. The most likely value of Kwas determined by
both choosing the smallest Kafter having reached a plateau
of the log probability of data Pr(X|K) values (Pritchard et al.
2009) and the Kstatistic of Evanno et al. (2005), with the
aid of STRUCTURE HARVESTER (Earl and vonHoldt,
2012).
Finally, gene flow was determined using Wright’s
(1951) equation, using the FST value: Nm =1–FST /4FST,
where Nm is the average number of migrants exchanged per
generation. The allozyme data were deposited in the genetic
diversity digest coded D-ALLOZ-48 in the Demiurge infor-
mation system (http://www.demiurge-project.org/).
Genetic analysis of RAPD data
All individuals were scored for the presence or absence of
RAPD fragments, and the data were entered into a binary
data matrix as discrete variables (1 for presence and 0 for
absence). Genetic diversity was estimated using Shannon’s
HSH (Lewontin 1972) and Nei’s h(1973) methods with
POPGENE v.1.32 (Yeh et al. 1997). Shannon’s diversity
index is frequently used in RAPD analyses because it is
insensitive to bias that may be introduced into data owing
to undetectable heterozygosity (Gustafson et al. 1999; Maki
and Horie 1999). Other measures for measuring the amount
of genetic variation were computed, such as Br (band rich-
ness, which can be defined as the number of bands expected
at each locus, and can be interpreted as an allelic richness
analogue, ranking from 1 to 2; Honnay et al. 2006) and
PLP (percentage of polymorphic loci), with the help of
the software AFLPDIV v1.1 (Coart et al. 2005). These two
measures of allelic richness were standardised to the low-
est sample size (n=8) by using the rarefaction approach
(El Mousadik and Petit 1996). Two additional measures of
allelic richness were obtained from the binary data matrix:
number of private fragments (fp, which are those bands
confined to one population), and number of rare frag-
ments (fr, those fragments occurring at low frequencies).
Fragments were considered rare when they occurred in
three or fewer individuals within the whole dataset (i.e. ≤
10% of the individuals).
As for the allozymes, the RAPD data were used to
examine the genetic structure of G. oleifolia by means
of an AMOVA and also with the Bayesian approach of
STRUCTURE 2.3.1 (Hubisz et al. 2009). The parame-
ters for the latter were set to the same values than those
for allozymes, and the program was also run in an analo-
gous way (10 independent runs of K=1–5). Gene flow
was calculated as for the allozymes (i.e. with Wright’s
(1951) equation). To generate similarity matrices, the data
were analysed by the Nei and Li coefficient (Nei and Li
1979). This algorithm calculates coefficients of similari-
ties based on the shared presence of bands and excludes
shared absence of bands as a criterion of similarity. A
neighbour-joining (NJ) tree was built to visualise the rela-
tionship among all the studied individuals using PAUP
4.0b10 (Swofford 2003). The RAPD data were deposited
in the genetic diversity digest coded D-RAPDS-52 in
the Demiurge information system (http://www.demiurge-
project.org/).
Results
Allozyme analysis
Among the 13 interpretable loci, we detected 22 alle-
les (data not shown). All of these alleles were exhibited
by the largest subpopulation (SAN), whereas the smaller
one (TOL) contained only 13 alleles (i.e. it was entirely
monomorphic). This finding, together with the fact that the
most common alleles for all the polymorphic loci at SAN
corresponded to those detected at TOL (data not shown),
can be attributed to the limited sample size for TOL or,
alternatively, to a larger incidence of genetic drift within
this much smaller subpopulation. Some of the alleles found
exclusively at SAN (Idh-1b,Idh-2a, and Mdh-1c) were rare,
occurring at very low frequencies (<0.05). The remaining
alleles found exclusively at SAN always occurred at fre-
quencies under 0.20. Of the polymorphic loci in SAN, two
(Idh-1 and Idh-2) exhibited four alleles, one (Mdh-1) three,
and the fourth (Pgm-2)two.
The only polymorphic subpopulation (SAN) showed
moderate to low values of genetic diversity (P=30.8%,
A=1.69, AR =1.41, and He=0.098). These values
were averaged with those of the monomorphic subpop-
ulation (TOL), and the resulting population-level genetic
variability for G. oleifolia would be considered to be very
low (Table 1): P=15.4%, A=1.34 and He=0.049. A
deficiency of heterozygotes at SAN was revealed by the
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Population genetics and conservation of Glandora oleifolia 505
Table 1. Summary of genetic diversity for the two sub-populations of Glandora oleifolia.
Allozymes1RAPD2
Sub-population PAA
pAR HoHeNo. loci Br3PLP3hH
SH
SAN 30.8 1.69 3.25 1.41 0.076 (0.145) 0.098 (0.167) 985 1.54 89.70 0.11 0.21
TOL 0.0 1.00 0.0 1.00 0.000 (0.000) 0.000 (0.000) 573 1.52 52.20 0.10 0.18
Population mean 15.4 1.34 1.62 1.21 0.038 0.049 779 1.53 70.95 0.11 0.19
Species level 30.8 1.69 3.25 1.69 0.067 0.089 1098 — — 0.11 0.21
Reference values for
endemic species (at
global scale)4,5
26.3 1.39 — — — 0.063 — — — 0.191 —
Reference values for
Mediterranean species626.4 1.41 — — 0.090 0.100 — — — — —
Reference values for
Mediterranean species
living in inland cliffs6
28.4 1.36 — — 0.092 0.104 — — — — —
1For allozymes: P, percentage of polymorphic loci; A, mean number of alleles per locus; Ap, mean number of alleles per polymorphic locus; AR, mean allelic richness (adjusted for a sample size
of eight individuals); Ho, observed heterozygosity; He, expected panmictic heterozygosity. Standard deviation in parentheses.
2For RAPD: Br, band richness; PLP, percentage of polymorphic loci (5%); h, Nei’s unbiased gene diversity; HST, Shannon’s index.
3Calculation is standardised to the lowest sample size (N=8).
4Hamrick and Godt (1990).
5Nybom and Bartish (2000).
6López-Pujol et al. (2009).
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506 A. del Hoyo et al.
Table 2. Estimates of F-statistics for all polymorphic
allozyme loci in the two sub-populations of Glandora oleifolia.
Locus FIS FST FIT
Idh-1 −0.019 0.076 0.058
Idh-2 0.153 0.125 0.260
Mdh-1 0.783 0.041 0.792
Pgm-2 0.282 0.089 0.346
Mean 0.226 0.093 0.298
FIS, inbreeding coefficient, FST, fixation index, and FIT, overall
inbreeding coefficient.
comparison of the value of observed heterozygosity (Ho)
with that of expected under panmixia (He). This deficiency
was also detected at the level of loci: the chi-square test
(χ2) showed that FIS values for three of the four polymor-
phic loci were significantly different from zero (P0.05)
and positive, indicating an excess of homozygotes (data
not shown). Moderate levels of inbreeding were also sup-
ported by the mean value of Wright’s inbreeding coefficient
(FIS =0.226; Table 2). Genetic divergence among subpop-
ulations was quantified by computing the FST parameter, a
measure of differentiation among subpopulations (Table 2).
The overall value of FST was relatively low (0.093) with
respect to the mean value of FIT (0.298), indicating that
only a small fraction of the genetic variability detected in
G. oleifolia is attributable to differences among subpopu-
lations. Accordingly, gene flow was estimated to be high
(Nm =2.44).
The AMOVA analysis showed that most of the genetic
variability of the species is found within subpopulations
(90.7%) and thus the genetic divergence between sub-
populations is relatively low (9.3%). The estimated log-
probability of data Pr(X|K) obtained with STRUCTURE
decreased progressively from K=1toK=5, which indi-
cates that all the individuals belong to a single genetic
cluster (Figure 2). The Kstatistic indicated K=2asthe
best grouping scheme, but it should be taken into account
that this method cannot evaluate K=1. In fact, the soft-
ware’s graphical output for K=2showedthatthetwo
geographic subpopulations did not match with the two
identified genetic clusters (Figure 2).
RAPD
The 10 primers used in this study rendered 1098 bands
for the 30 individuals analysed. The values of band rich-
ness (Br) and the percentage of polymorphic loci (PLP)
corrected for the sample bias were higher in SAN sub-
population compared with TOL (Table 1). In contrast to
allozymes, no (sub)population-specific (i.e. private) bands
were detected in the analysis (fpwas 0 for both subpopu-
lations), although the number of rare bands was very high
(fr=603 in SAN, which represented 54.9% of the total
number of bands detected for the species; and fr=285
in TOL, which accounted for 26.0%). The mean values of
genetic diversity at the population level according to the
Nei (h) and Shannon (HSH) indices were 0.11 and 0.19,
respectively. The SAN subpopulation showed higher values
of genetic diversity (h=0.11; HSH =0.21) than did TOL
(h=0.10; HSH =0.18).
In agreement with the results of the allozyme analysis,
the AMOVA confirmed that variation among subpopula-
tions contributed a mere 3.3%, whereas variation within
subpopulations contributed to up to 96.7% of the total
genetic variance of RAPD. Thus, the FST statistics based
on the AMOVA analysis indicated a very small genetic dif-
ferentiation among the two subpopulations (ST =0.033),
whereas the value of gene flow was very high (Nm =7.33).
Regarding the analysis carried out with STRUCTURE,
identical clustering schemes to those for allozymes were
found (K=1 using the log probability of data Pr(X|K),
and K=2 with the Kstatistic of Evanno; Figure 3).
Figure 2. Results of STRUCTURE for Glandora oleifolia based on allozyme data. The most likely Kis estimated (a) using the log
probability of data Pr(X|K) values (Pritchard et al. 2009); and (b) the Kstatistic of Evanno et al. (2005). (c) Assignation of individuals
to the genetic clusters at K=2. SAN, Sant Aniol d’Aguja; TOL, Toll de Monars.
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Population genetics and conservation of Glandora oleifolia 507
Figure 3. Results of STRUCTURE for Glandora oleifolia based on RAPD data. The most likely Kis estimated (a) using the log proba-
bility of data Pr(X|K) values (Pritchard et al. 2009); and (b) the Kstatistic of Evanno et al. (2005). (c) Assignation of individuals to the
genetic clusters at K=2. SAN, Sant Aniol d’Aguja; TOL, Toll de Monars.
The NJ tree of all individuals also showed that the individ-
uals only partially clustered according to their geographic
location: despite five individuals of TOL forming their
own (sub)cluster, the three remaining individuals from this
subpopulation linked together with individuals of SAN
subpopulation (Figure 4).
Discussion
Levels of genetic diversity in Glandora oleifolia
The two molecular markers used in this study (allozymes
and RAPD) both indicated low genetic variability at the
population level in G. oleifolia. The former, as anticipated
given the nature of the marker (Aagaard et al. 1998; Nybom
and Bartish 2000), showed lower levels of diversity than
the latter (He=0.049 for allozymes and h=0.11 for
RAPD). These values are slightly lower than those expected
for endemic species (reference values: He=0.063 with
allozymes, Hamrick and Godt 1990; He=0.191 with
RAPD, Nybom and Bartish 2000; Table 1), but consid-
erably lower than those usually found for mixed-mating
species (reference values: He=0.090 with allozymes,
Hamrick and Godt 1990; He=0.219 with RAPD, Nybom
and Bartish 2000). This relative lack of congruence indi-
cates that the genetic variability of plants does not always
depend only on the geographical range and the breed-
ing system, although both of these variables are generally
highly predictive (Hamrick and Godt 1996). Other factors,
including life-history, ecological and demographic charac-
teristics (e.g. the life strategy, seed dispersal mechanism,
habitat specificity, population sizes and growth rates) but
also evolutionary history (e.g. the occurrence of bottlenecks
and founder effects, the mode and timing of speciation) may
also be of paramount importance in shaping the genetic
variability of plant species (Karron 1987; Gray 1996; Booy
et al. 2000; López-Pujol et al. 2009). Thus, it is not rare
to find cases reporting high levels of genetic variation
Figure 4. Neighbour-joining tree of all the 30 studied individuals
of Glandora oleifolia with RAPD markers. Black squares are indi-
viduals from SAN sub-population, black circles are individuals
from TOL sub-population.
for rare and narrow endemic plants in the literature (e.g.
Gitzendanner and Soltis 2000; Fernández-Palacios et al.
2004; Godt et al. 2005).
As stated in the Introduction, many Mediterranean nar-
row endemics occur on cliffs and rocky outcrops, habitats
Downloaded by [UMA University of Malaga] at 02:14 17 January 2013
508 A. del Hoyo et al.
that are usually associated with low-stature and open vege-
tation. Other biological and ecological traits that are com-
mon to many Mediterranean narrow endemics are their
low investment in pollen and seed production (Lavergne
et al. 2004; Thompson 2005). These factors may enhance
inbreeding within populations, produce low recruitment
rates, prevent demographic growth, and limit gene flow
among populations. As endemics usually show small pop-
ulation sizes and their populations are highly isolated
(Barrett and Kohn 1991; Ellstrand and Elam 1993; Gaston
et al. 2000) an erosion of genetic diversity can be expected
(e.g. López-Pujol et al. 2009). This could be the case for
G. oleifolia, a species that exhibits most of the abovemen-
tioned traits. Its biological strategy, although still largely
unknown, seems to be close to that of rupicolous plants,
showing remnant population dynamics (as those of the
Tertiary relicts Borderea chouardii and Ramonda myconi),
in which longevity and population stability make recruit-
ment unnecessary for population persistence (Picó and Riba
2002; García 2003). The limestone-specific G. oleifolia is a
long-lived perennial shrub (longevity is enhanced by high
rates of clonal spread by stolons; A. del Hoyo, pers. obs.),
and some preliminary results have pointed out several seri-
ous reproductive constraints (such as pollen and pollinator
limitation and low seed production) as well as a low recruit-
ment rate (Ferrero 2006; Oliver 2008; Ferrero et al. 2011,
2012).
The evolutionary history of this palaeoendemic bor-
age (whose origin can be traced back to the late Miocene;
Ferrero et al. 2009; Weigend et al. 2009) may also account
for the depleted levels of genetic variability detected. The
outstanding role of Quaternary climatic oscillations (retreat
to glacial refugia/postglacial colonisation) in the current
genetic architecture of plant species is widely recognised
(e.g. Hewitt 1996; Petit et al. 2003; Thompson 2005; Hu
et al. 2009). In the Mediterranean Basin, cliffs, owing to
their relatively environmental stability and topographical
diversity (Tzedakis et al. 2002; Médail and Diadema 2009)
served as suitable refugia during the glacial/interglacial
Pleistocene cycles. Therefore, it is expected that rupi-
colous plants would retain high levels of genetic variabil-
ity (López-Pujol et al. 2009). Petrocoptis montsicciana
Bolòs & Rivas Mart. (Caryophyllaceae) and Ramonda
myconi may represent examples that confirm this pre-
diction, because relatively high levels of diversity found
with allozyme and RAPD markers, respectively, have been
reported for these species; this genetic wealth has been
attributed to the occurrence of multiple glacial refugia
and persistence of a large number of populations (López-
Pujol et al. 2001, J. López-Pujol and S. López-Viñallonga
unpubl. data; Dubreuil et al. 2008). However, depleted lev-
els of genetic diversity have also been reported for other
limestone plants such as Borderea chouardii or Erodium
rupestre (Cav.) Marcet (Geraniaceae) (Segarra-Moragues
and Catalán 2002, 2008; López-Pujol et al. 2006). For the
former, a combination of repeated population bottlenecks
and historical extinctions has been proposed to explain the
low genetic diversity found in the only extant population
(Segarra-Moragues and Catalán 2002, 2003). According to
the same authors (Segarra-Moragues and Catalán 2008),
reproductive and dispersal constraints could have prevented
any post-glacial expansion of the range of B. chouardii.
Similarly to B. chouardii, the occurrence of large demo-
graphic fluctuations during successive Quaternary cycles
(including strong bottlenecks during the most unfavourable
periods for the plant) would likely have increased genetic
drift within populations of G. oleifolia, thereby contribut-
ing to its current low levels of variability. The much
higher levels of genetic variation detected for the large
subpopulation (SAN) compared with the small one (TOL)
could be attributed to increased genetic drift operating in
the latter (as smaller populations are more prone to the
deleterious effects of genetic drift; Young et al. 1996).
Moreover, extinction and/or demographic decimation of
populations would have enhanced the species’ loss of
genetic diversity. This hypothesis cannot be discarded in
view of a possible scenario involving extensive glacia-
tion in the Pyrenees (Calvet 2004; Hughes et al. 2006)
(Figure 1). Remarkably, although G. oleifolia is the basal
species of the genus (Ferrero et al. 2009), it is also the
most geographically restricted. This characteristic of the
species probably reflects a hypothetical range contraction
throughout the Pleistocene. The extant population is located
outside of, but not far away (just a few kilometres) from
the nearest Pyrenean areas that were glaciated during the
Last Glacial Maximum (LGM) (the Canigou Massif; Calvet
2004; Figure 1). The advance of the glaciers during the
Pleistocene cold periods could have produced the extinction
of hypothetical former populations (presumably located
northwards and westwards, towards the Pyrenean summits,
at a higher elevation) and could have decimated others.
This scenario is supported by the recent extinction of two
additional subpopulations of the species, one on the French
(northern) side of the Pyrenees (Lamanère) and another on
the Spanish side (Beget) (Figure 1), which were present
until the late twentieth century (Sáez et al. 2010). Some
human disturbance would have been enough to cause the
extinction of previously decimated localities by the LGM
glacial advancement. Under this scenario, only those pop-
ulations located in warmer locations (at eastern, lower
altitudes with a southern exposure) could have managed to
survive. This is likely to have been the case for the only two
remaining subpopulations (which occur at altitudes below
1000 m a.s.l.). We should take into account that the low
mountain ranges in the south-easternmost corner of the
Pyrenees served as a refugium for numerous plant species
(Médail and Diadema 2009).
Conservation implications
We cannot quantify the effects of human disturbances on
the loss of genetic diversity at the species level, but this
loss may be huge (two of the four subpopulations of G.
oleifolia that occurred at the end of twentieth century are
now extinct). Regarding the current population, it should
be noted that Sant Aniol is one of the tourist hotspots
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Population genetics and conservation of Glandora oleifolia 509
of its region (Oliver 2008; A. del Hoyo pers. obs.); thus,
the significant human disturbance of the population could
also have contributed to the low levels of genetic diversity
observed in it. However, negative effects on genetic diver-
sity of this population might not yet be detectable because
these disturbances have occurred relatively recently and
because of the long lifespan of the species. These mod-
ern threats to G. oleifolia could also serve to increase the
genetic divergence among its two subpopulations, although
the amount of this divergence is still relatively low (the
value of FST obtained with allozymes and RAPD is below
0.010; moreover, the AMOVA, the Bayesian analysis of
genetic structure, and the NJ tree indicate that the two
subpopulations are genetically hardly distinguishable). The
proximity of these subpopulations (less than 5 km apart)
is probably maintaining sufficient gene flow, despite the
reproductive limitations observed in the species. Moreover,
some individuals have been observed to occur between the
two subpopulations (X. Oliver pers. obs.). These interme-
diate individuals could effectively connect the two subpop-
ulations.
The recent human disturbance (trekking, rock climb-
ing, canyoning) observed around the site where the species
occurs (Sáez et al. 2010; A. del Hoyo pers. obs.) could
be responsible for the disappearance of the species from
its northernmost and westernmost stations (Lamanère and
Beget). Moreover, competition with alien invasive species
is an additional risk factor. Invasive alien species detected
within the population include Buddleja davidii Franch.
(Loganiaceae), Erigeron karvinskianus DC. (Compositae),
and Fallopia baldschuanica (Regel) Holub (Polygonaceae)
(Oliver 2008; Sáez et al. 2010). The occurrence of inva-
sive alien species is of special significance because of
the low competitive ability often shown by the endemic
Mediterranean species, especially by those living in harsh
environments, such as rocky habitats (Médail and Verlaque
1997; Lavergne et al. 2003, 2004). To our knowledge,
some measures to eradicate the alien species have been
employed in the past (X. Oliver pers. comm.). However,
these measures have had only limited success.
Other in-situ as well as ex-situ conservation measures
are already in place. The two subpopulations are located
within the Alta Garrotxa Site of Community Importance
within the Natura 2000 network. This designation gives a
certain amount of protection to the plant and to its habi-
tat, although it is clearly insufficient since threats are still
in place (A. del Hoyo pers. obs.). In addition, the species
is listed as ‘vulnerable’ in the Catalogue of Protected
Flora of Catalonia (DOGC 2008). This listing implies the
need to elaborate a management plan. However, this has
not happened yet, which creates uncertainty regarding the
future viability of the species. Some monitoring has also
been carried out by volunteers (Oliver 2008), and fur-
ther field surveys will be made (X. Oliver pers. comm.)
to attempt to detect additional populations. Nevertheless,
in-depth demographic studies, which would reveal the
reasons for the low recruitment rates, have not yet been
carried out.
In ex-situ measures now underway, seeds are stored
and living plants are grown at the Marimurtra Botanic
Garden, near Barcelona. Germination tests carried out in
the same institution have shown very low germination rates.
Although more research on germination and recruitment
conditions is needed, it is highly advisable to sample addi-
tional individuals (in order to have seeds from as many
individuals as possible and to have a duplicate seed bank).
Given the lack of clear genetic differences between the two
supopulations, the seed collection could be centred on the
large subpopulation.
Acknowledgements
We thank Xavier Oliver and Victoria Ferrero for the data provided,
and Marc Calvet for granting us permission to reproduce the
map included in Figure 1. The comments of F.X. Picó (Associate
Editor) and two anonymous referees have led to great improve-
ments in the text. This study was funded by 2007ACOM00007
Catalan government grant. Jordi López-Pujol has benefited from
both a post-doctoral ‘JAE-Doc’ contract and a post-doctoral
‘Beatriu de Pinós’ fellowship (Generalitat de Catalunya).
Notes on contributors
Alberto del Hoyo’s research interests include phylogenetics and
biogeography of plants in Mediterranean climate regions
Jordi López-Pujol is interested in the conservation and biogeogra-
phy of plants, both in the Mediterranean Basin and in East Asia.
He has a long experience of working with allozymes.
Mi Yoon Chung is interested in ecology and conservation biol-
ogy of rare and endangered plant species in Korea, focusing on
orchids.
Blanca Lasso de la Vega works on the conservation of Iberian
endemic plants.
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