Patterns of genetic diversity in the highly threatened vascular flora of the Mediterranean Basin

Abstract

The Mediterranean Basin is widely recognized as one of the main plant biodiversity hotspots of the world. In its western part, the Catalan Countries, which represent the eastern section of the Iberian Peninsula, is one of the areas with the highest floristic diversity of the basin, containing more than 4,500 taxa, and showing a rate of endemism of about 5%. At the same time, this region is one of the most fragmented and manmodified of the basin. Here we present a synthesis of the literature concerning allozyme genetic diversity in plant taxa occurring in the Catalan Countries. From 36 taxa examined, which are mostly rare and/or threatened, relatively high levels of genetic variability have been detected (P = 28.85%, A = 1.46, and He = 0.112 for diploid taxa), although with a substantial percentage (around 6%) of rare alleles per population, high rates of imbreeding (FIS = 0.245), and a considerable genetic divergence among populations (GST = 0.248). The causes of such patterns, together with their relationship with different biological and ecological traits of the studied taxa, have been analyzed in detail. Such information has significant conservation implications, and may provide guidelines for setting up management and recovery plans of the Mediterranean endangered flora.

Full text

In: Endangered Species: New Research ISBN 978-1-60692-241-5
Editors: A. Columbus and L. Kuznetsov © 2009 Nova Science Publishers, Inc.
Chapter 2
PATTERNS OF GENETIC DIVERSITY IN THE HIGHLY
THREATENED VASCULAR FLORA OF THE
MEDITERRANEAN BASIN
Jordi López-Pujol1,*, Maria Bosch2, Joan Simon2 and
Cèsar Blanché2
1GReB, Botanic Institute of Barcelona (CSIC-ICUB), Passeig del Migdia s/n, 08038
Barcelona, Spain
2GReB, Laboratori de Botànica, Facultat de Farmàcia, Universitat de Barcelona, Avda.
Joan XXIII s/n, 08028 Barcelona, Spain
ABSTRACT
The Mediterranean Basin is widely recognized as one of the main plant biodiversity
hotspots of the world. In its western part, the Catalan Countries, which represent the
eastern section of the Iberian Peninsula, is one of the areas with the highest floristic
diversity of the basin, containing more than 4,500 taxa, and showing a rate of endemism
of about 5%. At the same time, this region is one of the most fragmented and man-
modified of the basin. Here we present a synthesis of the literature concerning allozyme
genetic diversity in plant taxa occurring in the Catalan Countries. From 36 taxa
examined, which are mostly rare and/or threatened, relatively high levels of genetic
variability have been detected (P = 28.85%, A = 1.46, and He = 0.112 for diploid taxa),
although with a substantial percentage (around 6%) of rare alleles per population, high
rates of imbreeding (FIS = 0.245), and a considerable genetic divergence among
populations (GST = 0.248). The causes of such patterns, together with their relationship
with different biological and ecological traits of the studied taxa, have been analyzed in
detail. Such information has significant conservation implications, and may provide
guidelines for setting up management and recovery plans of the Mediterranean
endangered flora.
* Author for correspondence
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Jordi López-Pujol, Maria Bosch, Joan Simon et al.
46
INTRODUCTION: BACKGROUND OF THIS STUDY - PATTERNS OF
GENETIC DIVERSITY IN RELATION TO ECOLOGY AND
EVOLUTIONARY TRAITS
In the last 25 years, numerous studies addressed to determine the existence of
relationships between genetic diversity of vascular plants and their biological, ecological and
geographical features have been published. These surveys were aimed to search –based on the
available bibliography– certain characteristics of plant species which allow to make
predictions on the levels and distribution of genetic variability of plant populations, given the
impossibility to perform genetic studies in all the endangered species or at least in a
significant fraction of them (Ellstrand & Elam, 1993; Hamrick & Godt, 1996a). On the other
hand, the present biodiversity crisis requires, in many occasions, to take decisions rapidly to
ensure the species conservation without having to wait to obtain the corresponding genetic
data (Spielman et al., 2004).
The first of these reviews was a collection of genetic (levels of diversity at population
scale) and ecological data (from 12 different traits) of a total of 113 plant taxa (Hamrick et al.,
1979). Some years later, this study was expanded up to 124 taxa and 14 ecological traits
(Loveless & Hamrick, 1984). The most comprehensive one is, however, Hamrick & Godt’s
review (1990), extensively cited in plant biology, which included genetic data from 449 taxa
at species level, at population level and among populations within the same species, and their
relationships with 8 large ecological categories. Later, the same authors (Hamrick & Godt,
1996b) performed an analysis with genetic data of approximately 1.500 entries (not
necessarily taxa, given that most of them were represented by more than one entry), and
comparing among several combinations of two ecological characteristics.
From all these studies, a series of conclusions with relevant implications for species
conservation were proposed. Despite the genetic variability depend on a network of
ecological, biological and also historical factors, usually intimately linked, there are two
characteristics that emerge over the remaining, because they explain a substantial part of the
variation of the genetic diversity of vascular plants: the geographical range and the breeding
system. Thus, species with narrow geographical ranges (endemic and/or rare species) often
present lower genetic diversity levels than those widespread, both at population and at species
scale, although there are not significant differences in the distribution of this diversity among
populations (Hamrick et al., 1979; Loveless & Hamrick, 1984; Hamrick & Godt, 1990). In
relation to the breeding system, the outcrossing species harbor more genetic variability than
the selfing ones, although the latter often exhibit a higher level of genetic differentiation
among populations (Loveless & Hamrick, 1984; Hamrick & Godt, 1990).
All these reviews have been criticized for the absence of a congruent statistical approach
(Karron, 1987; Gitzendanner & Soltis, 2000; Cole, 2003), given that the included taxa in
those analyses are considered as statistically independent data, ignoring their phylogenetic
relationships. This fact drives to a “pseudo-replication” phenomenon and to a lost of
statistical power of the analysis (Felsenstein, 1985; Silvertown & Dodd, 1996; Gitzendanner
& Soltis, 2000; Aguinagalde et al., 2005). For these authors, the best procedure is, in the
absence of phylogenetic approaches, limiting the comparisons to congeneric taxa, because
this will allow us to control the effects of phylogenetic inertia for the different ecological and
biological traits. An additional problem of these reviews of multiple species is the difficulty

Patterns of Genetic Diversity in the Highly Threatened Vascular Flora…
47
of standardization of the genetic data (such as different number of studied loci, different
interpretation of obtained phenotypes, different used programs for the calculation of genetic
variability parameters), a flaw that can be avoided in congeneric comparisons because data
from compared taxa usually come from the same publication (Gitzendanner & Soltis, 2000).
However, congeneric analyses exhibit the remarkably handicap that genetic data for only a
few pairs of congeneric taxa is available, limiting thus the power of this kind of approach.
Conversely, the classical compilations of multi-species genetic diversity allow the inclusion
of all genetic data available from literature, providing a more global approach to the patterns
of genetic diversity of plant species.
In the context of the comparisons between congeneric taxa, Karron (1987) was the first to
compare 11 pairs of rare and widespread congeneric plants in relation to their genetic
diversity and outcrossing rates; whereas he found that rare taxa showed lower levels of
genetic diversity than widespread ones, no significant differences were observed concerning
the outcrossing rates. More recently, Gitzendanner & Soltis (2000) performed a similar study
with a larger sample size (34 genera representing 102 taxa), comparing both the levels (at
species scale and at within population scale) and the distribution of genetic diversity,
obtaining a lower diversity for rare species but almost the same distribution of diversity
among populations. Lately, Cole (2003) included in his analysis a total of 247 plant species in
57 different comparisons of the genetic diversity between rare and common congeneric
species, with close results than those reported by Gitzendanner & Soltis (2000), in addition to
the finding of a lower genetic variability for the selfing species. Frankham (1995) was the
first in trying to correlate the threat degree of a given species and its levels of genetic
diversity. In his study, he compared the levels of genetic variation in 38 threatened/non-
threatened pair species (taxonomically related but not necessarily congeneric); in most of the
cases (32 pairs), the threatened species showed a lower genetic diversity, although only 6 of
38 pairs were plant species. In a more recent paper, Spielman et al. (2004) compared the
heterozygosity in 170 pairs of taxa (also taxonomically close) threatened/non-threatened,
from which 36 were plant species. From these, in 27 pairs the threatened taxa presented lower
heterozygosity than the non-threatened ones.
The studies consisting of comparisons of different characteristics among populations
belonging to the same taxon, such as population size or if they are insular or mainland
populations, are much more common. However, and despite these comparisons are reported
in numerous studies of genetic diversity of plant species, few are the approaches trying to find
a common pattern for plants and not only describing what occurs in a certain species. In the
review of Ellstrand & Elam (1993) concerning the genetic consequences of small population
size, from a list of 10 species, in 7 they reported a positive correlation between population
size and levels of genetic diversity. Analogously, Frankham (1996) found a positive
relationship between these two variables in 15 of 16 studied plant species. In a second work
from the same author (Frankham, 1997), mainland populations showed a significantly higher
genetic variation than insular ones in 8 of the 9 plant species included in the analysis.

Jordi López-Pujol, Maria Bosch, Joan Simon et al.
48
The Catalan Countries: A Representative Sample of the Mediterranean
Hotspot of Plant Biodiversity
The Mediterranean Basin is regarded as one of the 25 world hotspots of biodiversity
because of its enormous floristic richness (it harbors ca. 10% of the total amount of the higher
plants of the world in an area which accounts for only 1.6% of its surface) and exceptional
rates of endemism (13,000 of the 25,000 plant species occurring in the basin are endemics;
Médail & Quézel, 1997; Myers et al., 2000). These figures, which acquire special significance
when considering that the Mediterranean Basin is one of the most degraded regions of the
Earth (it retains less than 5% of its primary vegetation, Myers et al., 2000, and the total
number of threatened species is over 4,200; Greuter, 1991), have been attributed to a
complicated geological, climatic and biogeographical history (the basin is considered not only
a region of refuge and speciation, but also of floristic exchange) and to the large ecological,
geological and geographical heterogeneity of the basin (Verlaque et al., 1997; Médail &
Quézel, 1997; 1999; Blondel & Aronson, 1999; Thompson, 2005). The Mediterranean does
not constitute, however, a homogeneous centre of plant diversity; up to 10 smaller hotpots
(areas rich in species abundance and endemism) can be identified within the basin (Médail &
Quézel, 1997; 1999).
The ‘Catalan Countries’ is a geographical entity of ca. 70,000 km2 defined by those
territories in which the Catalan is the mother tongue, and is composed by one sovereign
country (Andorra), three autonomous regions of Spain (Catalonia, Valencia and Balearic
Islands), portions of a fourth one (Aragon), and the Department of Eastern Pyrenees, in
France (Fig. 1). It is a floristically very well studied territory, with regional floras published
as early as the second half of the 19th century (Costa, 1864; 1877; Barceló, 1879-1881), and a
flora for the whole territory completed in recent times (Bolòs & Vigo, 1984-2001). The
Catalan Countries, which account for only 2.9% of the land surface of the Mediterranean
Basin, contain 3,586 species or 4,604 taxa (including species, subspecies and varieties; Bolòs
et al., 2005), which represent ca. 15% of the floristic richness of this world hotspot (14.34%
at species level and 15.34% at taxon level). Thus, this European region is undoubtedly one of
the richest areas of the Mediterranean Basin in terms of vascular plant diversity. The mean
rate of endemism is, however, relatively low (around 5%), although several areas of the
Catalan Countries are included within some of the 10 Mediterranean ‘mini’ hotspots (see
Médail & Quézel, 1997), with rates of endemism close or even larger than 20% (Fig. 2). In
addition to their large taxonomic richness, the Catalan Countries also show a great diversity
of habitats and ecosystems, from coastal sand dunes and marshes to alpine meadows and
nival vegetation. In agreement with these data, the Catalan Countries should be considered as
a representative sample of the Mediterranean hotspots. A significant fraction of their plant
diversity is, however, currently threatened: although there is not a global figure for the
Catalan Countries, red lists available for several areas of this region (Laguna et al., 1998;
Sáez & Rosselló, 2001; Sáez et al., 2008) let us to think that the number of threatened taxa
may exceed 500.
The present work is aimed to explore the patterns of genetic diversity in vascular plants
from the Catalan Countries and, by extension, the Mediterranean Basin, with the following
specific goals: (i) to analyze the genetic data available from the literature concerning plant
species occurring in the considered geographic area (including those published by our team
during the last decade); (ii) to explore the relationships between several biological and

Patterns of Genetic Diversity in the Highly Threatened Vascular Flora…
49
ecological traits and the levels and distribution of genetic diversity; and (iii) to determine, on
one hand, if geological and climatic history (such as Messinian Salinity Crisis or Pleistocene
glaciations), and, on the other hand, if the continued impact of the human activities
(especially intense during the last century) have contributed, at least in some extent, to the
genetic diversity patterns found for this region.
0º 12º
12º 6º 18º6º
40º
48º
56º
0º 12º
12º 6º 18º6º
40º
48º
56º
Figure 1. Location of the Catalan Countries, in bold.
Figure 2. Centres of endemism in the Mediterranean Basin (Médail & Quézel, 1997). Several areas in
the Catalan Countries exhibit high rates of endemics, such as the Pyrenean and Pre-Pyrenean ranges,
the Catalonian interior ranges, the Balearic Archipelago, and Dianic ranges (the latter with taxes of
endemism over 20%). Reproduced from Médail & Quézel (1997) with permission.

Jordi López-Pujol, Maria Bosch, Joan Simon et al.
50
MATERIAL AND METHODS
Allozymes as Genetic Diversity Markers
Until the 1960s, the determination of genetic diversity in plant species relied on the
measure of morphometric traits. Studies based on these features presented several
disadvantages, such as these traits had quantitative variation (they were not discrete
phenotypic classes), they showed polygenic inheritance (i.e., the individual loci could not be
identified), their expression depended enormously on the environmental conditions, and
controlled crossings under homogeneous environmental conditions were required to
determine the inheritance mechanism of the studied traits (Allard et al., 1968; Berg &
Hamrick, 1997). Since the second half of the 1960, biochemical and molecular techniques
began to develop, which allowed, for the first time, the identification of alleles (i.e, qualitative
traits and not quantitative ones) in a relatively large sample of genetic loci. The basic
assumption in the use of these molecular markers consist in that genetic structure measured
with neutral or almost neutral genes (and subsequently independent on the environment) may
reflect evolving processes (e.g. inbreeding, genetic drift and gene flow) that affect the entire
genome (Bataillon et al., 1996; Hamrick & Godt, 1996a; Petit et al., 1998). The first
monogenic markers used were allozymes, still profusely employed nowadays. At the
beginning of the 1990s, with the development of the PCR technique (Polymerase chain
reaction), which allow the amplification of DNA fragments, a wide array of new types of
markers emerged, such as RFLPs, SNPs, AFLPs, ISSR and microsatellites.
Despite the development of all these new molecular techniques, allozymes still continue
being one of the most used markers in the detection of genetic diversity (Hogbin et al., 2000;
Ridgway, 2005) and possibly constitute the first election in conservation genetics (R.
Frankham, pers. com.) due to a series of advantages: technological (an optimum ratio cost-
effectiveness, the lack of necessity to have a previous knowledge of the genome, and the
simplicity of the technique), genetic (their inheritance is co-dominant, which allow to
discriminate phenotypically homozygotes from heterozygotes, and they are products of the
expression of coding regions of the genome, which permit a straightforward interpretation of
the obtained band patterns in genetic terms –alleles and loci–), and, as well, practical (there is
a large body of allozymic literature that, obviously, eases to perform comparisons). However,
they present some drawbacks compared to other molecular markers, such as the need of
samples coming from living tissues, the impossibility of automatization of the experimental
procedure, and the low level of polymorphism obtained, although often it is enough to detect
the genetic diversity at population scale (e.g. Crawford et al., 2001a; Ridgway, 2005).
Allozymes, therefore, continue being a perfectly valid research tool to study population
genetics of plant species, and they have been the selected marker to characterize the genetic
variability of the wild vascular flora from the Catalan Countries, that we present in this work.

Table 1. Genetic and ecological data for each of the 36 taxa included in the study
Taxon
Number of
populations
***
Number
of loci
% rare
alleles per
population**
P A Ho H
e F
IS
% private
alleles per
population
FSTa
or
GSTb
1 2 3 4 5 6 7 8 9 Reference
DIPLOID TAXA
1. Abies alba 23 (1) 10 0 40.0 1.40 — 0.063 — — — G W IM LP S O LD F NT Fady et al.
(1999)
2. Aconitum lycoctonum 19 (1) 10 — 29.0 1.50 0.093 0.100 0.066 — — D W M SP S O G F NT Utelli et al.
(1999)
3. Antirrhinum
pertegasii
4 (4) 14 — 17.3 1.21 0.050 0.080 — — 0.060b D E M SP S O G R T
(VU)*
Mateu-Andrés
(2004)
4. Antirrhinum
valentinum
5 (5) 21 — 23.8 1.33 0.184 0.075 — — 0.480b D E M SP S O G R T
(VU)*
Mateu-Andrés
& Segarra-
Moragues
(2000)
5. Borderea chouardii 1 (1) 21 4.17 9.5 1.14 0.078 0.046 -
0.461
— — D E M LP S O G R T
(CR)
Segarra-
Moragues &
Catalán (2002)
6. Borderea pyrenaica 6 (1) 21 0 19.0 1.24 0.135 0.085 -
0.500
— — D E M LP S O G R NT Segarra-
Moragues &
Catalán (2002)
7. Brassica montana 5 (3) 11 6.71 36.4 1.88 — 0.184 — 6.41 0.181b D W M SP — M G R NT Lázaro &
Aguinagalde
(1998)
8. Cheirolopus
intybaceus
5 (4) 10 1.92 15.8 1.18 — 0.247 — 5.05 0.293b D W IM SP — O — MS NT Garnatje et al.
(1998)
9. Cyclamen balearicum 28 (19) 9 0.87 24.2 1.30 0.006 0.062 0.948 1.01 0.112a D W IM LP S M LD F NT Affre et al.
(1997)
10. Delphinium bolosii 3 (3) 15 1.82 28.9 1.40 0.107 0.104 0.019 7.73 0.252a D E M SP AS M G MS T
(EN)
Orellana et al.
(2007)
11. Delphinium gracile 3 (3) 11 10.02 45.5 1.77 0.198 0.159 0.039 18.46 0.073a D W M A S M G MG NT Bosch (1999)
12. Delphinium
pentagynum subsp.
formenteranum
1 (1) 9 6.25 40.7 1.60 0.125 0.180 0.358 — — D E I SP AS O G MS T
(CR)
López-Pujol et
al. (2003a)
13. Delphinium
verdunense
2 (2) 11 16.11 40.9 2.15 0.154 0.205 0.451 17.31 0.028a D W M A — M G OT NT Bosch (1999)
14. Erodium rupestre 5 (5) 14 0 7.1 1.07 0.009 0.025 0.613 1.25 0.372a D E M LP S O G R NT López-Pujol et
al. (2006)

Table 1. Continued
Taxon Number of
populations
***
Number
of loci
% rare
alleles per
population**
P A Ho He FIS % private
alleles per
population
FSTa
or
GSTb
1 2 3 4 5 6 7 8 9 Reference
15. Hippocrepis
balearica
1 (1) 15 6.25 6.7 1.10 0.011 0.010 — — — D E I LP S O G R NT González-
Candelas &
Montolío (2000)
16. Hippocrepis grosii 1 (1) 15 6.25 20.0 1.30 0.122 0.101 — — — D E I LP S O G MC T
(EN)
González-
Candelas &
Montolío (2000)
17. Hippocrepis
valentina
12 (12) 15 5.78 24.4 1.27 0.099 0.085 -
0.179
3.35 0.171a D E M LP S O G R NT González-
Candelas &
Montolío (2000)
18. Juglans regia 15 (1) 15 3.85 53.3 1.73 0.331 0.365 0.091 — — D W IM LP S O LD F NT Fornari et al.
(1999)
19. Lolium rigidum 10 (2) 5 — — 3.90 0.408 0.514 — — — M W IM A — O G OT NT Oliveira & López
(1999)
20. Petrocoptis
montsicciana
4 (4) 16 15.06 70.3 2.20 0.121 0.239 0.486 6.30 0.376b D E M LP S O G R NT López-Pujol et al.
(2001)
21. Petrocoptis pardoi 3 (3) 16 11.76 56.3 1.90 0.072 0.192 0.620 8.56 0.354b D E M LP S O G R T
(VU)
López-Pujol et al.
(2001)
22. Pinus halepensis 6 (3) 15 5.26 26.7 1.27 0.059 0.064 0.068 0 0.031b G W IM LP S O LD F NT Agúndez et al.
(1997)
23. Pinus halepensis 15 (6) 5 — — — 0.262 0.228 0.128 — — G W IM LP S O LD F NT Agúndez et al.
(1999)
24. Pinus pinaster 12 (2) 18 — 41.7 2.00 — 0.159 — — — G W IM LP S O LD F NT Salvador et al.
(2000)
25. Pinus pinaster 32 (7) 14 — 51.4 1.71 — 0.103 0.038 — — G W IM LP S O LD F NT González-
Martínez et al.
(2005)
26. Pinus sylvestris 23 (1) 11 28.57 72.7 2.54 0.316 0.334 0.056 — — G W IM LP S O LD F NT Prus-Glowacki &
Stephan (1994)
27. Quercus suber 18 (2) 14 — 32.1 — 0.135 0.145 0.123 — — D W IM LP S O LD F NT Jiménez et al.
(1999)
28. Quercus suber 7 (1) 13 8.33 61.5 1.85 0.189 0.229 — — — D W IM LP S O LD F NT Elena-Rosselló &
Cabrera (1996)
29. Seseli farrenyi 3 (3) 14 31.09 83.3 3.00 0.120 0.297 0.592 4.41 0.041b D E M SP S O LD MC T
(EN)
López-Pujol et al.
(2002)

TAXON Number of
populations
***
Number
of loci
% rare
alleles per
population**
P A Ho He FIS % private
alleles per
population
FSTa
or
GSTb
1 2 3 4 5 6 7 8 9 Reference
30. Silene diclinis 2 (2) 26 3.45 5.8 1.11 — 0.023 — 3.33 0.048b D E M SP S O G MG T
(EN)
Prentice (1984)
31. Silene hifacensis 8 (8) 12 0.89 7.3 1.08 — 0.085 — 0.96 0.575b D E IM SP S O — MC T
(VU)*
Prentice et al.
(2003)
32. Silene sennenii 5 (5) 21 7.90 20.9 1.31 0.049 0.063 0.253 0 0.271a D E M SP AS — LD MG T
(EN)
López-Pujol et al.
(2007a)
33. Stachys maritima 5 (5) 20 2.72 14.0 1.16 0.065 0.066 -
0.102
0.95 0.316b D W M SP AS O G OT T
(CR)
López-Pujol et al.
(2003b)
POLYPLOID TAXA
34. Delphinium
montanum
7 (7) 15 12.78 23.8 1.48 0.075 0.082 0.089 2.51 0.135b D E M SP S O G OT T
(VU)
López-Pujol et al.
(2007b)
35. Medicago sativa
(wild)
9 (2) 5 — 90.0 3.10 — 0.255 — — — D W M SP — O — MG NT Jenczewski et al.
(1999)
36. Thymus loscosii 8 (6) 5 18.40 83.3 2.94 0.474 0.423 -
0.125
1.04 0.025b D W M SP AS O G MS NT López-Pujol et al.
(2004)
1. Taxonomic group: G: gymnosperms; D: dicots; M: monocots.
2. Geographical range: E: endemic (present in less than 50 localities or in an area lower than 10,000 km2); W: widespread (widespread in the Catalan Countries and even extending to
nearby territories).
3. Insularity: I: only insular distribution (of analyzed populations); M: only mainland distribution; IM: insular + mainland distribution.
4. Life strategy: A: annual plants (include the biannual ones); SP: short-lived perennials (usually < 10 years); LP: long-lived perennials (usually >10 years).
5. Mode of reproduction: AS: plants with clonal capacity (asexual + sexual reproduction); S: sexual.
6. Breeding system: O: outcrossing; M: mixed strategy (outcrossing + selfing).
7. Seed dispersal mechanism: G: gravity (short distance); LD: long distance strategies (epizoocory, endozoocory, anemocory, hydrocory and others).
8. Habitat: MC: maritime cliffs; MS: Mediterranean-type shubland; MG: meadows and grasslands; R: inland cliffs and rock exposures; F: forests; OT: others.
9. Threat status: T: threatened (listed in one of the IUCN categories); NT: non-threatened. The assignment of threat categories has been done using the most recent criteria of IUCN
(2001).
*1994 IUCN categories.
** For the calculation only the populations with N>10 have been considered, given that populations with smaller size cannot contain alleles with a frequency lower than 0.05.
*** In parenthesis, number of populations located in the Catalan Countries.

Jordi López-Pujol, Maria Bosch, Joan Simon et al.
54
Compilation of Allozyme Studies in Taxa of the Vascular Flora from Catalan
Countries
In order to characterize the genetic diversity of the wild vascular flora from the Catalan
Countries, a search of the available literature on allozymic diversity of taxa (species or
subspecies) located totally or at least partially in this geographic area (Fig. 1) has been carried
out. The available genetic data of the collected plant taxa has been used to build a database, to
which the body of data on the genetic variation obtained by our research team during the last
decade has been added.
Some of the included taxa, such as Pinus halepensis, Pinus pinaster and Quercus suber,
are object of more than one study. In these cases, and following Hamrick & Godt’s (1996b)
recommendations, they should be considered as independent entries and, thus, calculating the
means of the genetic parameters for each species with multiple entries should be avoided. The
population size, the number of populations and the number of assayed loci are traits that often
vary substantially depending on the research team that carry out the investigation, and
sometimes, each work represents a different region of the distribution area of a given taxon.
Thus, in these cases we should consider that each entry reports a unique information and,
given the multi-taxon database size (three cases of 36 entries in total; Table 1), the
redundancy –consequence of the multiple entries for certain taxa–, has a negligible effect in
the mean values of the different genetic parameters (cf. Hamrick & Godt, 1996b). We only
have taken into account data from wild flora species; this fact explains the no inclusion, for
instance, of the flagship Mediterranean endemics Lysimachia minoricensis (at present extinct
in the wild), because all the individuals analyzed electrophoretically had their origin in
botanical gardens (Ibáñez et al., 1999).
In addition, we have only considered the surveys which included the genetic
interpretation of the electrophoretic band patterns. In the taxa in which monomorphic as well
as polymorphic loci were obtained (or only polymorphic), we extracted (or calculated in the
case of taxa for which one or more parameters lacked, provided always the raw genetic data,
i.e. genotypic and/or allelic frequency matrices, were available) the parameters that describe
the intrapopulation genetic diversity levels: A (the mean number of alleles per locus), P (the
percentage of polymorphic loci when the most common allele had a frequency below 0.95),
Ho (the observed heterozygosity), He (the expected panmictic heterozygosity), and FIS (the
inbreeding coefficient). When the allelic frequencies were available, we also calculated the
percentage of rare alleles (those in proportions lower than 0.05) per population, and the
percentage of private alleles per population. We also extracted (or calculated) one indicator of
the distribution of genetic variability between populations: FST (the fixation index) or,
alternatively, GST (the coefficient of genetic differentiation), considering that both are
equivalent (in fact they show the same value when there are two alleles per locus; Hartl &
Clark, 1989) in order to increase the sample size. For all the cases in which the calculation of
one or more genetic parameters has been required, two of the commonest computer programs
in population genetics were used: BIOSYS-1 (Swofford & Selander, 1989) and GENESTAT-
PC (Whitkus, 1988).
Summarizing, we have built a database with the values of the different genetic
polymorphism parameters at population level for 36 plant taxa (Table 1). Nevertheless, the
number of existing studies on allozyme diversity of vascular flora from the Catalan Countries
is slightly higher. The plant taxa for which genetic diversity parameters at population level

Patterns of Genetic Diversity in the Highly Threatened Vascular Flora…
55
(or, alternatively, the raw genetic data necessary for their calculation) were neither provided
in the original publications nor obtained by direct request from the authors, were not taken
into account for this work. Consequently, these were not included in the database and are the
following: Antirrhinum intermedium, A. latifolium, A. litigiosum, and A. majus (Mateu-
Andrés & de Paco, 2005), Quercus ilex (Michaud et al., 1995; Toumi & Lumaret, 2001), and
Q. coccifera (Toumi & Lumaret, 2001).
Allozymic Diversity and Biological and Ecological Traits of the Collected
Taxa
Taking into account the reviews published until the present moment (Hamrick et al.,
1979; Loveless & Hamrick, 1984; Hamrick & Godt, 1990, 1996b), we selected several
biological and ecological traits with a significant influence in the levels and distribution of the
genetic diversity. All plant taxa from the multi-taxon database have been classified in one of
the following categories for each ecological trait: (1) the large taxonomic group
(gymnosperms, dicotyledons or monocotyledons); (2) the geographical range (endemic or
widespread); (3) the insularity (only insular distribution, only mainland distribution or both
insular and mainland distribution); (4) the life strategy (annuals, short-lived perennials or
long-lived perennials); (5) mode of reproduction (a mixed asexual and sexual strategy or only
sexual); (6) the breeding system (outcrossing or mixed strategy), (7) the seed dispersal
mechanism (by gravity or a long distance mechanism), (8) the habitat (maritime cliffs,
Mediterranean-type shrubland, meadows and grasslands, inland cliffs and rock exposures,
forests and others); and (9) the threat status (threatened or non-threatened). A last trait has
been added, the ploidy level (10), which allow the comparison of genetic variability between
diploid and polyploid species. The criteria for including the collected plant taxa within each
category for the different ecological traits as well as the meaning of the used abbreviations are
detailed in the footnote of Table 1. The information used to classify each taxon in the
corresponding categories has been obtained from the original publications (i.e. genetic
diversity surveys), other papers focused in other aspects of these taxa (for instance, the
breeding systems), several local floras (such as Flora dels Països Catalans; Bolòs & Vigo,
1984-2001), red books or other types of publications. For all the taxa included in the multi-
taxon database, we calculated the means (weighted by the product of the number of loci
analyzed and the number of studied populations) and standard deviations of the different
genetic parameters (A, P, Ho, He, FIT, FST or GST, the percentage of rare alleles per population
and the percentage of private alleles per population) for each category of the different
ecological traits abovementioned. To check the differences among the different categories of
each ecological trait we used the non parametric test of Kruskal-Wallis, given that in many
parameters the values were not normally distributed.
Secondly and when it was possible, we have also performed comparisons between
congeneric taxa, as suggested by Karron (1987), Gitzendanner & Soltis (2000) and Cole
(2003). Nevertheless, given the small sample sizes, we were not able to use any of the proper
statistical tests to develop this kind of analyses (paired-data tests), and comparisons are thus
only qualitative. We have only carried out comparisons with a selection of ecological traits
(geographical range, insularity, threat status and ploidy level) because the congeneric taxa in
our dataset often belong to the same category for most of the traits. Finally, we have

Jordi López-Pujol, Maria Bosch, Joan Simon et al.
56
completed the study with the comparison among populations within the same taxon: from our
multi-taxon database, we have selected all the taxa which have populations belonging to
different categories for certain traits (insularity and threat status). All statistical calculations
have been performed with STATGRAPHICS Plus 5.0 program.
RESULTS AND DISCUSSION
Levels and Distribution of Genetic Diversity in Vascular Plant Species from
the Catalan Countries
In Table 1 is compiled allozyme data of vascular flora from the Catalan Countries which,
together with the data reported by the authors of the present contribution, consist of a total of
137 populations, belonging to 33 diploid taxa and 3 polyploid ones. One general conclusion
derived from this analysis is that mean values of genetic diversity for diploid vascular plants
from the Catalan Countries are apparently poor at population level (P = 28.85%, A = 1.46, He
= 0.112; Table 2) when comparing with the results from other reviews of genetic diversity.
For example, in the first compilation of genetic variability data done by Hamrick et al. (1979),
values of genetic diversity computed from a pool of 113 taxa were higher (P = 36.8%, A =
1.69, He = 0.156) than those presented here. However, in a posterior review with a larger
sample size (653 entries representing 449 species), Hamrick & Godt (1990) reported
somewhat lower values (P = 34.2%, A = 1.53, He = 0.113), close to the obtained values in this
study. The value of expected heterozygosity, the parameter which better describes the genetic
variability (Berg & Hamrick, 1997), is almost the same in this analysis than in Hamrick &
Godt’s (1990) one. Moreover, a series of considerations concerning these results should be
taken into account: (i) the taxa included in our analysis represent a very limited geographical
area –the Catalan Countries– in comparison to the aforementioned reviews, in which taxa
from all latitudes were considered; (ii) an important part of the species collected here (about
half of the total) were originally studied, precisely, because of its endemic and/or threatened
character (conditions that usually implies a lack of genetic variability of these taxa;
Gitzendanner & Soltis, 2000; Cole, 2003; Spielman et al., 2004); (iii) we have only selected
the populations of species located geographically within the Catalan Countries boundary;
thus, for some of the collected taxa only one population is included in our analysis, cases
which are usually not considered in the general reviews; and (iv) the number of both
gymnosperms and monocots in this compilation is very small, despite these are taxonomical
groups often exhibiting high levels of genetic diversity (see Hamrick & Godt, 1990).
Therefore, it may be more proper to talk of levels of moderate or relatively high genetic
diversity in the studied vascular plants from the Catalan Countries, as expected in a region
which should have acted as a biological diversity reservoir or refuge (for both species and
genetic diversity) during the Quaternary glacial periods (Hewitt, 1996; 1999; Taberlet et al.,
1998; Thompson, 2005), but which also suffered a drastic transformation of its original
ecosystems since the Holocene (Blondel & Aronson, 1999; Thompson, 2005).
The levels of genetic diversity for the studied taxa by our research team during the last 10
years show a great variability (Table 1): from very high values, such as those unexpectedly
found in Seseli farrenyi (P = 83.3%, A = 3.00, He = 0.297; Fig. 3), a narrow endemic species

Patterns of Genetic Diversity in the Highly Threatened Vascular Flora…
57
with only three small subpopulations amounting only about 2,000 individuals (López-Pujol et
al., 2002), or the tetraploid Thymus loscosii (P = 85.0%, A = 3.00, He = 0.422), to extremely
low levels of variability, such as those obtained in Stachys maritima (P = 14.00%, A = 1.16,
He = 0.066; Fig. 3) and in Erodium rupestre (P = 7.1%, A = 1.07, He = 0.025; Fig. 3).
Nevertheless, these reported values fall well within the range found for the reviewed plant
taxa from the Catalan Countries: P vary from 90.0% in Medicago sativa to 5.8% in Silene
diclinis; the interval of A is from 3.90 in Lolium rigidum to only 1.08 in another species of
Silene, S. hifacensis; and last but not least, the range of expected heterozygosity (He) is also
wide: from 0.514 in Lolium rigidum to 0.010 in Hippocrepis balearica. This large disparity of
values for the different basic parameters of diversity in the vascular plants from the Catalan
Countries is not surprising given the large variability in the autoecologic traits and
phylogenetic origins of the different taxa included in this review. In addition, the very
complicated geological and biogeographic history of this Mediterranean region, as well as
occurred throughout the basin, has no doubt involved the existence of many different
evolutionary paths and processes (Thompson, 2005) giving place to a wide array of genetic
diversity patterns for vascular plant species.
Table 2. Genetic diversity parameters for the different categories of each ecological
trait of the plant taxa included in this study
Category Number
of taxa
% rare
alleles per
population1
P A Ho H
e F
IS
% private
alleles per
population
FSTa or
GSTb
(1) TAXONOMIC GROUP
NS NS NS NS NS NS — —
Gymnosperms 6 8.34
(15.20)
44.70
(19.48)
1.69
(0.57)
0.163
(0.182)
0.130
(0.106)
0.061
(0.015)
0 0.031
Monocots 1 — — 3.90 0.408 0.514 — — —
Dicots 26 5.74
(7.04)
26.60
(20.79)
1.44
(0.46)
0.085
(0.074)
0.109
(0.091)
0.280
(0.394)
3.61
(5.63)
0.255
(0.165)
(2) GEOGRAPHICAL RANGE
NS * * * * NS NS NS
Endemic 16 6.87
(8.11)
26.40
(23.52)
1.41
(0.52)
0.090
(0.049)
0.100
(0.081)
0.212
(0.389)
3.28
(3.03)
0.291
(0.179)
Widespread 17 4.30
(8.35)
30.93
(16.35)
1.62
(0.69)
0.098
(0.124)
0.131
(0.124)
0.319
(0.289)
3.76
(7.78)
0.164
(0.119)
(3) INSULARITY
NS NS NS NS NS NS NS NS
Insular 3 6.25
(0.00)
19.66
(17.13)
1.29
(0.25)
0.080
(0.065)
0.084
(0.085)
0.358 — —
Insular +
mainland
13 2.56
(9.45)
30.03
(19.92)
1.47
(0.81)
0.094
(0.140)
0.126
(0.139)
0.448
(0.328)
1.33
(2.24)
0.248
(0.241)
Mainland 17 7.45
(8.50)
28.54
(22.26)
1.46
(0.52)
0.093
(0.053)
0.105
(0.080)
0.138
(0.402)
4.42
(5.97)
0.247
(0.154)
(4) LIFE STRATEGY
NS NS NS NS NS NS NS NS
Annual 3 12.45
(4.31)
43.66
(3.25)
2.23
(1.14)
0.215
(0.136)
0.229
(0.193)
0.204
(0.291)
18.00
(0.81)
0.055
(0.032)
Short-lived
perennial
12 5.95
(9.37)
22.15
(20.73)
1.37
(0.53)
0.097
(0.045)
0.102
(0.083)
0.148
(0.254)
2.55
(2.80)
0.300
(0.180)
Long-lived
perennial
18 3.97
(7.66)
33.29
(21.63)
1.48
(0.44)
0.080
(0.104)
0.112
(0.104)
0.286
(0.420)
2.90
(3.37)
0.205
(0.151)

Jordi López-Pujol, Maria Bosch, Joan Simon et al.
58
Table 2. Continued
Category Number
of taxa
% rare
alleles per
population1
P A
Ho
He FIS
% private
alleles per
population
FSTa or
GSTb
(5) MODE OF REPRODUCTION
NS NS NS NS NS NS NS NS
Asexual +
sexual
4 2.56
(2.88)
21.70
(11.47)
1.28
(0.18)
0.068
(0.035)
0.075
(0.054)
0.079
(0.211)
1.77
(4.21)
0.285
(0.033)
Sexual 25 5.95
(9.25)
30.62
(22.55)
1.47
(0.51)
0.095
(0.094)
0.109
(0.098)
0.289
(0.394)
3.42
(5.81)
0.243
(0.195)
(6) BREEDING SYSTEM
NS NS NS NS NS NS NS NS
Outcrossing 27 6.38
(8.81)
29.32
(22.90)
1.47
(0.68)
0.108
(0.105)
0.119
(0.121)
0.108
(0.357)
3.05
(2.73)
0.285
(0.186)
Mixed strategy 5 3.74
(6.25)
29.74
(8.67)
1.49
(0.35)
0.065
(0.082)
0.102
(0.059)
0.643
(0.437)
5.66
(7.48)
0.130
(0.089)
(7) SEED DISPERSAL MECHANISM
NS * NS NS NS NS * NS
Gravity (short
distance)
19 5.69
(4.94)
26.49
(17.99)
1.44
(0.66)
0.101
(0.091)
0.105
(0.114)
0.092
(0.390)
5.13
(6.11)
0.255
(0.154)
Other
mechanisms
(long distance)
12 7.28
(12.17)
37.03
(20.38)
1.60
(0.57)
0.079
(0.119)
0.119
(0.112)
0.421
(0.311)
0.99
(2.09)
0.140
(0.111)
(8) HABITAT
NS NS NS NS NS NS NS NS
Maritime cliffs 3 9.75
(16.11)
29.41
(40.71)
1.63
(1.05)
0.120
(0.001)
0.145
(0.118)
0.592 2.01
(2.44)
0.412
(0.377)
Mediterranean-
type shrubland
3 2.29
(2.53)
22.10
(12.45)
1.20
(0.21)
0.110
(0.013)
0.155
(0.071)
0.075
(0.240)
5.79
(1.89)
0.243
(0.029)
Meadows and
grasslands
3 7.05
(3.35)
21.04
(20.04)
1.33
(0.34)
0.085
(0.105)
0.069
(0.070)
0.202
(0.151)
4.12
(9.84)
0.175
(0.122)
Inland cliffs 10 6.57
(5.22)
28.40
(21.37)
1.36
(0.40)
0.092
(0.057)
0.104
(0.077)
0.127
(0.536)
4.34
(2.87)
0.283
(0.149)
Forests 11 3.27
(10.61)
36.30
(16.14)
1.53
(0.40)
0.084
(0.121)
0.115
(0.108)
0.438
(0.308)
0.80
(0.71)
0.095
(0.057)
Others 4 5.13
(9.47)
18.85
(19.02)
1.42
(1.39)
0.106
(0.178)
0.123
(0.229)
-0.002
(0.391)
3.90
(11.57)
0.264
(0.204)
(9) THREAT STATUS
NS NS NS NS NS NS NS NS
Non-
threatened
21 5.31
(7.78)
32.77
(19.26)
1.52
(0.67)
0.092
(0.120)
0.126
(0.124)
0.277
(0.354)
4.06
(6.82)
0.183
(0.137)
Threatened 12 6.67
(8.84)
23.68
(22.81)
1.49
(0.54)
0.095
(0.042)
0.094
(0.077)
0.177
(0.391)
2.67
(3.40)
0.310
(0.191)
(10) PLOIDY LEVEL
NS NS NS NS NS NS NS NS
Diploid 33 5.88
(8.04)
28.85
(20.86)
1.46
(0.63)
0.094
(0.101)
0.112
(0.112)
0.245
(0.357)
3.47
(5.62)
0.248
(0.159)
Tetraploid 3 14.03
(3.97)
40.67
(36.44)
1.89
(0.89)
0.164
(0.282)
0.164
(0.170)
0.041
(0.151)
2.18
(1.04)
0.110
(0.078)
1For the calculation only the populations with N>10 have been considered, given that populations
with smaller size cannot contain alleles with a frequency lower than 0.05.
* p < 0.05; ** p < 0.01; *** p < 0.001; NS: non-significant, according to Kruskal-Wallis test.
In parenthesis, standard deviation.
One of the parameters poorly considered in the extant reviews of genetic diversity is the
percentage of rare alleles (i.e. those present in very small proportion, lower than 0.05%) per

Patterns of Genetic Diversity in the Highly Threatened Vascular Flora…
59
population. Rare alleles are of notably importance in conservation genetics, especially for the
management strategies (both in-situ and ex-situ) of rare and endangered plant species,
because these alleles may have a significant adaptative or evolutive value (Caujapé-Castells
& Pedrola-Monfort, 2004; Gapare et al., 2005). This parameter can give an idea of the effect
of genetic drift within the plant populations (for example, a population that does not contain
rare alleles probably could have suffered the eroding effects of genetic drift), and might be
used as an indicator of the real threat that a population is facing (e.g. a high percentage of rare
alleles means that a considerable fraction of the genetic variability of that population can be
lost in few generations in the case of reductions in its size, fragmentation or significant
demographic fluctuations). In the populations of diploid vascular plants from the Catalan
Countries, nearly 6% of the total number of alleles are rare (Table 2), which indicates that a
non-negligible amount of the genetic variation is facing a great and imminent risk of loss.
Thus, populations highlighting for their large amount of rare alleles should be targeted for
special attention by the biodiversity management administrations, probably constituting
conservation units (CUs) and thus meriting specific monitoring. It is remarkably the case of
some species, such as Pinus sylvestris and Seseli farrenyi, where almost the third part of all
their alleles are in frequencies lower than 0.05 (see Fig. 4).
Figure 3. Some of the studied endemic and/or threatened plant taxa from the Catalan Countries. a.
Petrocoptis montsicciana; b. Delphinium montanum; c. Erodium rupestre; d. Stachys maritima; e.
Seseli farrenyi.
[Pictures: a., b. and d., Jordi López-Pujol, c. Joan Simon, e. Maria Bosch]
e
abc
dee
aa bb cc
dd

Jordi López-Pujol, Maria Bosch, Joan Simon et al.
60
0
5
10
15
20
25
30
35
Borderea pyrenaica
Erodium rupestre
Abies alba
Cyclamen balearicum
Silene hifacensis
Delphinium bolosii
Cheirolopus intybaceus
Stachys maritima
Silene diclinis
Juglans regia
Bord erea choua rdii
Hippocrepis valentina
Pinus halepensis
D. pentagynum subsp. formenteranum
Hippocrepis balearica
Hippocrepis grosii
Brassica montana
Silene sennenii
Quercu s su ber
Delphinium gracile
Petrocoptis pardoi
Petrocoptis montsicciana
Delphinium verdunense
Pinus sylvestris
Seseli farrenyi
Rare alleles (%)
0
5
10
15
20
25
30
35
Borderea pyrenaica
Erodium rupestre
Abies alba
Cyclamen balearicum
Silene hifacensis
Delphinium bolosii
Cheirolopus intybaceus
Stachys maritima
Silene diclinis
Juglans regia
Bord erea choua rdii
Hippocrepis valentina
Pinus halepensis
D. pentagynum subsp. formenteranum
Hippocrepis balearica
Hippocrepis grosii
Brassica montana
Silene sennenii
Quercu s su ber
Delphinium gracile
Petrocoptis pardoi
Petrocoptis montsicciana
Delphinium verdunense
Pinus sylvestris
Seseli farrenyi
Rare alleles (%)
Figure 4. Percentage of rare alleles per population in the species from the Catalan Countries included in
the study.
The second of the processes that drive to a genetic diversity erosion, together with
genetic drift, is the increase of consanguinity or inbreeding rates, phenomenon that usually
occurs when the size of populations decreases. The most used parameter in population
genetics to measure the increase of consanguinity is the inbreeding coefficient (FIS; Wright,
1951), which measures the deviation from Hardy-Weinberg equilibrium (i.e. the effects of
non-random mating) within populations by comparing observed and expected heterozygosity.
For the vascular plants from the Catalan Countries, the value obtained for this parameter was
significantly different of zero and positive (FIS = 0.245; Table 2), which indicates that the
populations of the studied plants are relatively inbred. The small population sizes reported for
many of the plant taxa included in this review (sometimes under the minimum viable
population [MVP] size requirements) and the subsequent forced biparental inbreeding may
account for this. Habitat degradation, in terms of fragmentation and reduction of the
population sizes, or in terms of decrease of the quality and quantity of the pollinator services,
has also probably contributed to the levels of consanguinity detected in the plant species from
the Catalan Countries.
A significant fraction of genetic diversity of the studied taxa from this Mediterranean
region is due to differences among populations: the interpopulation genetic divergence is
considerable (GST = 0.248; Table 2), slightly higher than that reported by Hamrick & Godt
(1990; GST = 0.224). Moreover, the Catalan plant taxa also exhibit a noticeable amount of
private alleles in their populations (ca. 3.5%; Table 2), which also indicates divergence
among populations. Patterns of marked genetic differentiation have been reported for species
with populations located in different glacial refugia of Southern Europe (Iberian Peninsula,
Italy and the Balkans), and they have been attributed to the isolation of populations in these
favorable places during the Quaternary glacial cycles (e.g. Hewitt, 1996; 1999; 2000; Petit et

Patterns of Genetic Diversity in the Highly Threatened Vascular Flora…
61
al., 2003; 2005; Thompson, 2005). This isolation associated with climatic oscillations even
produced speciation events in the Mediterranean basin, such as those described for the genus
Senecio to be occurred during the last 1 My (Comes & Abbott, 2001). However, as evidenced
in phylogeographical studies performed in recent years (e.g. Olalde et al., 2002; Gómez et al.,
2005; López de Heredia, 2007; Dubreuil et al., 2008), species also exhibit a high degree of
interpopulation genetic differentiation within the large proposed refugia (i.e. a pattern of
‘refugia-within-refugia’; see Gómez & Lunt, 2007, for a complete review for the case of the
Iberian Peninsula). The very high levels of genetic divergence found in several species
included in the present review, such as Erodium rupestre and the two Petrocoptis species (ca.
40%; Table 1 and Fig. 3) may respond to this pattern. These Tertiary taxa are today restricted
to several rocky massifs, locations which probably served as isolated (allopatric) refugia
during the Pleistocene glacial cycles (Fig. 5).
Figure 5. In the Mediterranean Basin, rocky habitats contain many endemics, partly due to their role as
refugia during the Quaternary. On the left, schistose maritime cliff in Cape Creus (NE Catalonia); on
the right, limestone cliffs in the Pre-Pyrenean ranges (N Catalonia).
[Pictures: Núria Membrives (left) and Maria Bosch (right)]
Intraspecific (among populations) genetic divergence, however, might also predate the
onset of the Ice Ages (see Taberlet et al., 1998; Hewitt, 2000; Petit et al., 2005; Gómez &
Lunt, 2007; Magri et al., 2007) in the case of the relict species such as the above examples.
Thus, other previous major geological events occurred in the Mediterranean and entirely
affecting the Catalan Countries, such as the Messinian Salinity Crisis (occurred during the
Late Miocene), the Mediterranean microplate movements (from Late Oligocene to Pliocene)
or even the Alpine orogeny (back to Oligocene) cannot be discarded as factors shaping
current patterns of genetic structure of populations.
A much more recent episode, however, might have contributed to the remarkable genetic
differentiation of plant populations in the Catalan Countries: the destruction and
fragmentation of habitats taken place in this region as consequence of human activities. These
processes may have several genetic consequences on the plant populations, such as genetic
drift and inbreeding, which imply a reduction of intrapopulational genetic variation, and the
interruption of gene flow, that drives to an increase of divergence among populations (Young
et al., 1996; Oostermeijer et al., 2003). Despite reliable data on fragmentation are not
available for the Catalan Countries, its effects are evident in the littoral and peri-urban areas
(Fig. 6). As an illustrative example, Stachys maritima, a species growing in coastal dunes, has
lost more than 90% of its original habitat (López-Pujol et al., 2003b; Fig. 3) due to

Jordi López-Pujol, Maria Bosch, Joan Simon et al.
62
urbanization. From the observation of herbarium specimens, it can be deduced that this
species had a more o less continuous distribution along the coast at the beginning of the
twentieth century; today, the remaining patches are genetically very divergent (above 30%;
Table 1). Fragmentation is, however, more moderated in the interior of the territory, where
even in some zones an increase of patch connectivity is occurring due to abandonment of
plenty of agriculture lands during recent decades, phenomenon also detected in France
(Lavergne et al., 2006).
Figure 6. Changes in land use in tourist locations in the Catalan Countries have driven to extensive
habitat destruction and fragmentation (mainly due to building and urbanization in previously natural
zones) in the last century. As a consequence, many sensitive plant species (such as those growing in
sand dune habitats) have become rarer and threatened, loosing some of their genetic diversity. Beach of
the village of Pals (Girona province, Catalonia): on the left, before transformation (1910); on the right,
in recent times (2003), where the threatened species Stachys maritima (Lamiaceae) is found.
[Pictures: Cèsar Blanche personal archive (left) and Cèsar Blanché (right)]
Genetic Diversity and Ecological Characteristics
1. Taxonomic group. In the present review, the basic parameters of diversity give higher
values for monocots, followed by gymnosperms and, finally, dicots (Table 2). The
dicotyledons from the Catalan Countries (26 taxa in total) contain levels of diversity (P =
26.60%, A = 1.44, He = 0.109) very close or even higher than those found for dicots in the
survey of Hamrick & Godt (1990; P = 29.0%, A = 1.44, He = 0.096) which –as previously
stated– suggest an overall high genetic variation for the plant species of this Mediterranean
region. The genetic divergence between populations for dicots from the Catalan Countries is
also noticeable (25.5%), of the same order than in the review of Hamrick & Godt (1990;
27.3%).
2. Geographical range. It has been postulated that genetic variability is higher in species
with broad distribution areas than in geographically restricted species. Several papers
comparing values of genetic diversity between endemic and widespread distributed species
found significant differences for parameters A, P, Ho, and He (Karron, 1987; Hamrick &
Godt, 1990; Gitzendanner & Soltis, 2000; Cole, 2003). In contrast, there are no differences
between endemics and widespread species for inbreeding coefficient (Cole, 2003) or genetic
differentiation among populations (Hamrick & Godt, 1990; Gitzendanner & Soltis, 2000). In
the present survey, endemic species exhibit lower levels of genetic diversity than widespread
ones, with significant differences for all the analyzed descriptors of genetic variability (A, P,

Patterns of Genetic Diversity in the Highly Threatened Vascular Flora…
63
Ho, and He, see Table 2). It should be noted that the endemic species from the Catalan
Countries show a higher proportion of rare alleles per population and a higher divergence
between populations (GST = 0.291 vs. 0.164, Table 2) than those which are widespread,
although, in such cases, differences are not significant.
Table 3. Genetic diversity parameters for the two categories of each ecological trait in
congeneric taxa. References for each taxon can be found in Table 1
Taxon Category
% rare alleles
per
population*
P A Ho H
e F
IS
% private
alleles per
population
FSTa or
GSTb
(1) GEOGRAPHICAL RANGE
Delphinium bolosii Endemic 1.82 28.9 1.40 0.107 0.104 0.019 7.73 0.252a
Delphinium
pentagynum subsp.
formenteranum
Endemic 6.25 40.7 1.60 0.125 0.180 0.358 — —
Mean (endemic) 2.71 30.9 1.43 0.110 0.117 0.075 7.73 0.252
Delphinium gracile Widespread 10.02 45.5 1.77 0.198 0.159 0.039 18.46 0.073a
Delphinium
verdunense
Widespread 16.11 40.9 2.15 0.154 0.205 0.451 17.31 0.028a
Mean
(widespread)
12.46 43.7 1.92 0.180 0.177 0.204 18.00 0.055
(2) INSULARITY
Delphinium
pentagynum subsp.
formenteranum
Insular 6.25 40.7 1.60 0.125 0.180 0.358 — —
Delphinium bolosii Mainland 1.82 28.9 1.40 0.107 0.104 0.019 7.73 0.252a
Delphinium gracile Mainland 10.02 45.5 1.77 0.198 0.159 0.039 18.46 0.073a
Delphinium
verdunense
Mainland 16.11 40.9 2.15 0.154 0.205 0.451 17.31 0.028a
Mean (mainland) 7.67 37.0 1.69 0.147 0.144 0.121 13.38 0.144
Hippocrepis
balearica
Insular 6.25 6.7 1.10 0.011 0.010 — — —
Hippocrepis grosii Insular 6.25 20.0 1.30 0.122 0.101 — — —
Mean (insular) 6.25 13.3 1.20 0.066 0.055 — — —
Hippocrepis
valentina
Mainland 5.78 24.4 1.27 0.099 0.085 -0.179 3.35 0.171a
(3) THREAT STATUS
Borderea chouardii Threatened 4.17 9.5 1.14 0.078 0.046 -0.461 — —
Borderea pyrenaica Non- threatened 0 19.05 1.24 0.135 0.085 -0.500 — —
Delphinium bolosii Threatened 1.82 28.9 1.40 0.107 0.104 0.019 7.73 0.252a
Delphinium
pentagynum subsp.
formenteranum
Threatened 6.25 40.7 1.60 0.125 0.180 0.358 — —
Mean (threatened) 2.71 30.9 1.43 0.110 0.117 0.075 7.73 0.252
Delphinium gracile Non-threatened 10.02 45.5 1.77 0.198 0.159 0.039 18.46 0.073a
Delphinium
verdunense
Non- threatened 16.11 40.9 2.15 0.154 0.205 0.451 17.31 0.028a
Mean (no
threatened)
12.46 43.7 1.92 0.180 0.177 0.204 18.00 0.055
Hippocrepis grosii Threatened 6.25 20.0 1.30 0.122 0.101 — — —
Hippocrepis
balearica
Non-threatened 6.25 6.7 1.10 0.011 0.010 — — —
Hippocrepis
valentina
Non-threatened 5.78 24.4 1.27 0.099 0.085 -0.179 3.35 0.171a
Mean (no
threatened)
5.82 23.0 1.26 0.092 0.079 -0.179 3.35 0.171a
Petrocoptis pardoi Threatened 11.76 56.3 1.90 0.072 0.192 0.620 8.56 0.354b
Petrocoptis
montsicciana
Non-threatened 15.06 70.3 2.20 0.121 0.239 0.486 6.30 0.376b

Jordi López-Pujol, Maria Bosch, Joan Simon et al.
64
Table 3. Continued
Taxon Category
% rare
alleles per
population*
P A Ho H
e F
IS
% private
alleles per
population
FSTa or
GSTb
(4) PLOIDY LEVEL
Delphinium bolosii Diploid 1.82 28.9 1.40 0.107 0.104 0.019 7.73 0.252a
Delphinium gracile Diploid 10.02 45.5 1.77 0.198 0.159 0.039 18.46 0.073a
Delphinium
pentagynum subsp.
formenteranum
Diploid 6.25 40.7 1.60 0.125 0.180 0.358 — —
Delphinium
verdunense
Diploid 16.11 40.9 2.15 0.154 0.205 0.451 17.31 0.028a
Mean (diploid) 7.55 37.3 1.68 0.145 0.147 0.134 13.38 0.144
Delphinium
montanum
Tetraploid 12.78 23.8 1.48 0.075 0.082 0.089 2.51 0.135b
*For the calculation only the populations with N>10 have been considered, given that populations with
smaller size cannot contain alleles with a frequency lower than 0.05.
The fact that endemic species showed rates of genetic divergence higher than those of
more widespread distribution can be due to the typology of endemicity in the Mediterranean
Basin (and, by inclusion, in the Catalan Countries). Following Lavergne et al. (2004),
Mediterranean endemic species, in contrast with those of broad distribution, produce fewer
flowers and these are smaller, they show a reduced separation between stigmas and anthers, a
reduced P/O ratio and a lower seed production. This reduced pollen and seed transfer
capabilities may limit the opportunities of gene flow, thus favoring a higher genetic
differentiation between populations of endemic species. In fact, values of genetic divergence
for endemic species from the Catalan Countries (GST = 0.291) are higher than those reported
for rare/endemic species in other reviews of genetic diversity (0.206 in Gitzendanner &
Soltis, 2000; 0.212 in Cole, 2003). Regarding congeneric taxa comparisons, the only genus
with available data coming from both endemic and widespread species is Delphinium,
showing higher values of genetic variability for species of broad distribution (Table 3).
3. Insularity. Insularity is widely considered as a factor with an important influence on
levels and distribution of genetic diversity of vascular plants because of two biogeographic
characteristics shared by islands: a restricted geographic area and their isolation (Rieseberg &
Swensen, 1996). Plants inhabiting in islands usually show, in general, a noticeable genetic
depauperation caused by several factors: i) bottlenecks associated to founder effects; ii) small
population size of island populations, favoring genetic drift and inbreeding, and iii) adaptation
to island ecosystems, that can imply a loss of dispersal ability and a reduced resistance to
predators and diseases (Frankham, 1998, Crawford et al., 2001b).
Levels of genetic variability for exclusively insular taxa from the Catalan Countries, that
is, those from Balearic Islands (P = 19.66%, A = 1.29, He = 0.084) are lower than those for
mainland taxa (P = 28.54%, A = 1.46, He = 0.105) and than those with both insular and
mainland populations (P = 30.03%, A = 1.47, He = 0.126), although these differences are not
significant, and we should take into account the small sample size of the insular taxa (N = 3)
in our database (Table 2). For the species of exclusively Balearic distribution, these values are
slightly higher than those found for plant taxa from other archipelagos: Hawaii (P = 25.00%,
A =1.32; He = 0.064; DeJoode & Wendel, 1992) and Juan Fernández (P = 21.00%, He =
0.044; Crawford et al., 2001b), but not for the Canary Islands (He = 0.137; Francisco-Ortega
et al., 2000, see Fig. 7). The recognized role as glacial refugium for the Balearic Islands

Patterns of Genetic Diversity in the Highly Threatened Vascular Flora…
65
(López de Heredia, 2005; Médail, 2007) may provide an explanation for the not very
depauperate levels of genetic variation found in the plant taxa of this archipelago.
Unfortunately, data on interpopulational divergence for exclusively Balearic taxa are not
available. Concerning congeneric comparisons, while for Hippocrepis insular species show
lower diversity, for Delphinium the results are the opposite (Table 3). At population level,
island populations of Cheirolophus intybaceus and Pinus halepensis contain lower genetic
variability than mainland populations. In contrast, and contrary to the expected rule, island
populations of Silene hifacensis display higher diversity than mainland ones (Table 4), a
result which can be interpreted as a consequence of the severe degradation of its habitat and
excessive herborization by botanists in the Alacant seashore, in the mailand (Prentice et al.,
2003).
Figure 7. Expected heterozygosis levels (He) for the insular species from different archipelagos.
4. Life strategy. The vascular plants from Catalan Countries showing the highest levels
of genetic diversity are annuals, with values (expressed as percentage of polymorphic loci,
mean number of alleles per locus and observed and expected heterozygosity) higher than
those for perennial taxa, either long-lived or short-lived (see Table 2); however, differences
are not statistically significant. These results could be surprising, if compared with those
reported by Hamrick et al. (1979), in which long-lived perennials showed the highest levels of
genetic diversity. Moreover, annual plants from the Catalan Countries show lower levels of
genetic divergence among populations than in perennials, the contrary of the results given by
Hamrick and collaborators (Loveless & Hamrick, 1984; Hamrick & Godt, 1990). For these
authors, long-lived perennial species show the highest intrapopulation genetic diversity levels
because they usually display a combination of ecological characteristics favoring the
maintenance of genetic variability: cross-pollination, high fecundity and a higher dispersal
capability both for pollen and seeds, factors which, in addition, limit the genetic divergence
among populations (Hamrick & al., 1979; Loveless & Hamrick, 1984). The small sample size
of the annuals in our database (N = 3), besides the fact that these all have a widespread
distribution, may have determined these unexpected results for the Catalan plant taxa.
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0,16
Hawaii Juan Fernández Canary Islands Balearic Islands
Expected heterozygosity
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0,16
Hawaii Juan Fernández Canary Islands Balearic Islands
Expected heterozygosity

Jordi López-Pujol, Maria Bosch, Joan Simon et al.
66
Table 4. Genetic diversity parameters for the two categories of each ecological trait in
populations of one taxon. The values of the genetic parameters are population means.
References for each taxon can be found in Table 1
Taxon Category
Number
of loci
Number of
populations
% rare
alleles per
population*
P A Ho H
e
(1) INSULARITY
Silene hifacensis Insular populations 12 6 1.19 9.7 1.11 — —
Mainland populations 12 2 0 0 1.00 — —
Cheirolophus intybaceus Insular populations 12 1 0 10.0 1.10 — 0.346
Mainland populations 10 3 2.56 17.8 1.24 — 0.213
Pinus halepensis Insular populations 15 1 10.53 13.3 1.27 0.056 0.055
Mainland populations 15 2 2.63 23.3 1.27 0.061 0.069
Pinus halepensis Insular populations 5 1 — — — 0.196 0.199
Mainland populations 5 5 — — — 0.234 0.275
(2) THREAT STATUS
Delphinium bolosii Threatened populations 15 2 4.54 26.7 1.40 0.095 0.097
Non-threatened populations 15 1 0 33.3 1.30 0.115 0.117
Delphinium montanum Threatened populations 15 3 13.67 20.0 1.44 0.064 0.075
Non-threatened populations 15 4 12.21 25.0 1.48 0.083 0.087
Erodium rupestre Threatened populations 14 3 0 7.1 1.07 0.013 0.019
Non-threatened populations 14 2 0 7.1 1.07 0.003 0.034
Petrocoptis montsicciana Threatened populations 16 2 7.99 65.6 1.95 0.136 0.232
Non-threatened populations 16 2 22.13 75.0 2.40 0.105 0.246
Seseli farrenyii Threatened populations 14 1 22.22 78.6 2.60 0.124 0.285
Non-threatened populations 14 2 35.52 85.7 3.20 0.118 0.303
Silene sennenii Threatened populations 21 3 9.33 19.0 1.32 0.046 0.055
Non-threatened populations 21 2 3.33 23.8 1.31 0.055 0.095
Thymus loscosii Threatened populations 5 3 19.23 93.3 3.16 0.481 0.441
Non-threatened populations 5 5 20.16 80.0 2.90 0.466 0.410
*For the calculation only the populations with N>10 have been considered, given that populations with
smaller size cannot contain alleles with a frequency lower than 0.05.
5, 6, and 7. Mode of reproduction, breeding system and seed dispersal mechanism.
The reproduction systems sensu lato as well as the geographical range have been postulated
as major determinants of both levels and distribution of genetic diversity in vascular plants
(Hamrick & Godt, 1990; Hamrick & Godt, 1996b). The mode of reproduction (sexual or
sexual + asexual), however, does not have a significant effect on the patterns of genetic
variation in plants (Hamrick & Godt, 1990). For the Catalan species, the taxa with clonal
capacity (i.e. those combining asexual and asexual strategies) show genetic diversity levels
slightly lower than those exclusively sexual, although these differences have no statistical
significance (Table 2).
Breeding systems, in contrast, have a marked effect on the levels and distribution of
genetic diversity in plants. Selfing plants are characterized by a much lower amount of
intrapopulation genetic diversity and much more pronounced interpopulation divergence than
outcrossing (Hamrick & Godt, 1990; Hamrick & Godt, 1996b). In the present survey on
genetic diversity of the Catalan Countries, we do not have allozyme data on strictly selfing
species, and the comparison between mixed strategy species (combining rates of selfing and
outcrossing) and strictly outcrossing show that these latter have somewhat higher
intrapopulation genetic diversity, although differences are not significant (Table 2). In
relation to the genetic divergence among populations, outcrossing taxa exhibit unexpectedly a
higher value, although differences, again, are not statistically significant.

Patterns of Genetic Diversity in the Highly Threatened Vascular Flora…
67
Concerning the seed dispersal mechanism, species dispersing their seeds by gravity (i.e.
seeds are deposited close to the parental plants) usually exhibit a lower intrapopulational
diversity and a higher interpopulational divergence compared to species dispersing seeds by
wind, water or animals (Loveless & Hamrick, 1984; Hamrick & Godt, 1990). In the vascular
plants from the Catalan Countries, taxa dispersing seeds by gravity displayed, as expected,
lower levels of intrapopulational genetic diversity and higher levels of interpopulational
divergence than those with long-distance dispersal systems, although differences were only
significant for the percentage of polymorphic loci and for the percentage of private alleles per
population (Table 2).
8. Habitat. According to Lavergne et al. (2004), Mediterranean endemic species are
often characterized by growing in sites with high rock cover, pronounced slopes, low-stature
open vegetation, and few coexisting species. In fact, more than 70% of the endemic species
reviewed in this study (12 of 17, see Table 1) grow in rocky habitats (Fig. 5), in most cases
corresponding to calcareous rocky outcrops of middle altitude/high mountain ranges (e.g.
Erodium rupestre and Petrocoptis spp.; Fig. 3), whereas some of the endemic taxa are located
in maritime cliffs, such as Silene hifacensis or Seseli farrenyi (Fig. 3). Only a single species
growing in rocky habitats reviewed in this survey have a widespread distribution (Brassica
montana). This close relationship between rocky habitat and endemicity is not new to science:
it has already been proven not only for several areas of the Catalan Countries, such as
Catalonia (a recent review of Catalan endemisms revealed that c. 70% of endemic species
from this area grow in rocky habitats; Sáez et al., 2008), Valencia (Laguna et al., 1998), and
the Balearic Archipelago (Alomar et al., 1997), but also for other regions of the
Mediterranean Basin, e.g. Corsica (Médail & Verlaque, 1997), SE France (Médail &
Verlaque, 1997; Lavergne et al., 2004), Italian Maritime Alps (Casazza et al., 2005), the
Balkans (Stevanović et al., 2003), and Greece (Strid & Papanicolaou, 1985), and even in
other latitudes (e.g. Simmons et al., 1998; Wolf, 2001; Clements et al., 2006).
Species from both types of rocky habitats, especially seashore cliffs, show relatively high
levels of genetic diversity, but, at the same time, very high genetic divergence among
populations (Table 2). The flora growing in meadows and grasslands is the poorest
genetically, whereas taxa growing in forests show the lowest rates of variability distributed
among populations. The relatively high levels of genetic variation and the large genetic
divergence among populations detected in the plant taxa growing in rocky habitats may
respond to the widely recognized role of cliffs (especially those maritime and those
calcareous with a southern exposure) as glacial refugia (see the previous section of the
Discussion), which enabled both the survival of Tertiary lineages and episodes of population
differentiation and speciation, due to their relatively environmental stability and
topographical diversity (Küpfer, 1974; Chytrý et al., 2003; Riba et al., 2003; Lavergne et al.,
2004; Thompson, 2005). Many rocky endemics from the Catalan Countries show a high
persistence ability driven by their biological characteristics (high longevity, low recruitment
levels and poor long-distance dispersal), such as the taxa belonging to the genera Borderea
and Petrocoptis. These traits would have allowed the conservation of, at least, a substantial
part of their original genetic variability and maintained the strong population genetic structure
shaped by the Pleistocene climatic oscillations.
9. Threat status. Theoretically, it is expected that threatened species will present lower
levels of genetic variability than non-threatened species, because of the negative effects of
threats (in particular of anthropogenic origin) on plant populations, mainly consisting in the

Jordi López-Pujol, Maria Bosch, Joan Simon et al.
68
reduction of population size and/or their fragmentation, which drive to a loss of allelic
richness and to increased rates of inbreeding (Ellstrand & Ellam, 1993; Frankham, 1995;
Oostermeijer et al., 2003). In the present survey, although differences are not statistically
significant (see Table 2), the mean values for the different parameters of genetic diversity are
higher in the non-threatened taxa than in the threatened ones (considering threatened those
listed in any of the three risk categories established by the IUCN: CR, EN or VU). Looking at
data of congeneric comparisons, for three of the four genera for which data are available,
threatened taxa exhibit less genetic variability than those non-threatened (see Table 3).
Regarding the distribution of genetic diversity among populations, differences are also
detected: threatened species show higher interpopulation genetic divergence than those non-
threatened. It seems reasonable to suppose that habitat fragmentation (one of the main effects
derived from human activities in the Catalan Countries in recent times) have had some
influence in such figure, as fragmentation has a clear effect of gene flow disruption among
populations.
Figure 8. Comparison of the expected heterozygosity levels between threatened and non-threatened
populations of different taxa included in the study.
At population level comparisons, from the 7 species in which the threat status for each
studied population is known, in 6 species threatened populations show lower genetic diversity
levels than non-threatened populations (see Fig. 8 and Table 4). Only for Thymus loscosii, a
polyploid species, diversity levels for threatened populations are higher than those non-
threatened. Therefore, from the overall results it can be hypothesized that the threats affecting
plant species in the Catalan Countries could be responsible, at least in part, of a loss of
genetic variation in their natural populations (in the same way as reported by Spielman et al.,
2004) but also of an increase of their genetic divergence.
10. Ploidy level. The last biological characteristic analyzed here, ploidy level, has usually
a significant effect on genetic variability of vascular plants (Soltis & Soltis, 1989). Diploid
taxa from the Catalan Countries, as expected, show lower genetic diversity than polyploid
taxa: fewer polymorphic loci, fewer alleles per locus, and lower heterozygosity (both
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0,4
0,45
0,5
T NT T NT T NT T NT T NT T NT T NT
Delphinium bolosii
Delphinium montanum
Erodium rupestre
Petrocoptismo
ntsicciana
Seseli farrenyi
Silene sennenii
Thymus loscosii
Expected heterozygosity
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0,4
0,45
0,5
T NT T NT T NT T NT T NT T NT T NT
Delphinium bolosii
Delphinium montanum
Erodium rupestre
Petrocoptismo
ntsicciana
Seseli farrenyi
Silene sennenii
Thymus loscosii
Expected heterozygosity

Patterns of Genetic Diversity in the Highly Threatened Vascular Flora…
69
observed and expected), but higher degree of inbreeding and higher genetic divergence
among populations (Table 2). Polysomic inheritance of polyploids has an effect of reduction
of the negative effects of bottlenecks and genetic drift, and is responsible as well of the high
heterozygosity levels currently detected in polyploids (Soltis & Soltis, 2000; López-Pujol et
al., 2004), which buffers the effects of inbreeding. The observed differences here on the
interpopulation genetic differentiation between diploids and polyploids (the latter exhibit
lower genetic divergence among populations) have a more uncertain explanation. The
perennial habit, a high incidence of vegetative propagation, and a better colonizing ability
respect to diploids, phenomena strongly linked to the ecological success of polyploids, could
be responsible in part of this finding.
CONCLUSION
Patterns of Genetic Diversity in the Flora from the Catalan Countries
The data compiled in the present study review the knowledge on allozyme diversity of
the wild flora from the Catalan Countries since the middle of the 1980s (the first published
genetic survey of a plant species native to this region corresponds to Silene diclinis; Prentice,
1984) up to the present time. The present work, of course, is not the only source of data
available for genetic diversity of plant species in this geographical area (those surveys using
PCR-based molecular markers are not included here), but the relatively large body of
allozymic literature (not still available for other genetic markers) allow for approaches to the
patterns of diversity and their possible causes, based in numerical comparisons. Using the
current available knowledge, thus, we are making a first evaluation of the levels and
distribution of genetic diversity of this Mediterranean region, which is the primary objective
of this work.
In relation to the levels of genetic variability, the values found for diploid taxa are
comparable to those reported at global scale (Hamrick & Godt, 1990). Considering the
methodological aspects previously stated (see the previous section), levels of diversity should
be considered as moderate or relatively high, which are explained, among other reasons, by
the role played by this region as a glacial refuge of flora. Nevertheless, nearly 6% of the total
number of alleles within populations occur in frequencies lower than 0.05, suggesting that a
significant part of genetic variability can be lost in few generations at the present rate of
habitat loss and fragmentation. The inbreeding levels for the taxa reviewed are relatively
high, partly also attributable to fragmentation and reduction of population sizes. Genetic
divergence between populations is also high, a common feature for plant species inhabiting
Mediterranean glacial refuges, but which could have been enhanced by the human-induced
habitat destruction and fragmentation.
When comparing levels and distribution of genetic variation among different categories
of the biological and ecological studied traits, it should be pointed out that endemic species
show a lower genetic variability but a higher divergence among populations than widespread
ones; that strictly insular species exhibit some degree of genetic depauperation; that
outcrossing species present higher genetic diversity than those with mixed strategies; that
species with gravity dispersed seeds display lower diversity but more divergence among

Jordi López-Pujol, Maria Bosch, Joan Simon et al.
70
populations than those with long-distance dispersal mechanisms; that rupicolous species show
levels of genetic diversity relatively high, but their populations are very divergent, and,
finally, that diploids have lower polymorphism than polyploids. One of the new contributions
of this work is that, altogether with the ‘classic’ ecological traits, the threat status is presented
as a predictive factor of genetic variability of vascular plants. Threatened taxa are less diverse
genetically but, at the same time, more divergent (among populations) than those non-
threatened, giving support to the hypothesis of Spielman et al. (2004) of a true and verifiable
genetic impact of threats on plant species.
However, the low statistical significance in many of the comparisons presented in this
study recommends the need to add new data of genetic diversity for a higher number of
species, which will enable further advances in understanding the genetic variability and their
distribution in the vascular flora from the Catalan Countries. A higher sample size for certain
categories should allow to check some anomalous results obtained in this analysis (e.g. that
annual species showed higher genetic variability than perennials). The incorporation of new
taxa should make possible, on the other hand, to perform new comparisons, both congeneric
and intraspecific (among populations), for a higher number of biological and ecological traits.
In the framework of pair comparisons (species and populations), it should be useful to include
genetic data from other molecular markers different of allozymes (AFLP, RFLP,
microsatellites, and so on). In this sense, we confidently expect the full launching of the
SAGE software developed by Caujapé-Castells (2007) for genotype analysis, which runs with
distinct molecular marker data sources. Finally, it is convenient to note that the study of
additional biological characteristics could provide new points of view useful for interpreting
patterns of genetic variability in plant taxa. The determination of the absolute age of a given
taxon (that is, if it is relict or of recent origin), in addition to explore its effect on the levels
and distribution of genetic diversity, can be used for the delimitation of endemicity centers,
understood as glacial refuge of flora (with high concentrations of paleoendemisms) or as
centers of differentiation of flora (amounting many neoendemisms).
CONSERVATION IMPLICATIONS
Some recent diagnosis on the conservation status of the vascular plants from the Catalan
Countries reviewed under the scope of the present contribution (Laguna et al., 1998; Sáez &
Rosselló, 2001; Bañares et al., 2003; Carrillo et al., 2008; Sáez et al., 2008) advised about the
endangered situation of an important part of this flora, a worrisome situation also occurring in
other Mediterranean regions (e.g. Raimondo et al., 1994; Montmollin & Iatrou, 1995;
Fennane & Ibn Tattou, 1999; Jeanmonod et al., 2001; Çakan et al., 2005). Changes in land
use, overexploitation and urbanization activities mainly associated to tourism development
have lead to a continous habitat loss and/or fragmentation, increasing the number and level of
threatened species. As showed in the previous paragraphs, a moderate but detectable genetic
diversity loss begins to emerge as a feature both for endemic as well as for widespread
species growing in the Mediterranean Basin.
Together with new efforts to carry out new research on genetic diversity markers, the
existing recorded diversity can be applied directly to drive actions oriented to the
conservation and/or recovery of endangered species and populations (Avise & Hamrick,

Patterns of Genetic Diversity in the Highly Threatened Vascular Flora…
71
1996; Frankham et al., 2002). Among the main indications of the genetic diversity surveys,
these constitute essential tools in the sampling strategy and conservation priorities of ex-situ
initiatives, as well as in the monitoring of translocation or population reinforcement practices
(Young & Murray, 2000; Schoen & Brown, 2001; Schaal & Leverich, 2004; Schwartz et al.,
2006; Liu et al., 2006). The absence of data concerning genetic variability and population
structure have lead in the past to unsuccessful conservation and restoration actions (although
expensive), such as that reported for Lysimachia minoricensis. This species endemic to the
Catalan Countries (restricted to the island of Menorca, in the Balearic Archipelago), was
reintroduced in the wild from botanical garden materials but with no genetic diversity (Ibáñez
et al., 1999), which probably produced the complete loss of all transplanted individuals. The
detailed study of the genetic diversity (with allozymes or other molecular markers)
constitutes, fortunately, the backbone of many conservation plans (both in-situ and ex-situ)
launched in recent times in the Catalan Countries. Examples include the reintroduction plan
of Delphinium bolosii in a location extinct in 1912, with the goal of reproducing the genetic
structure of an extant population (Blanché et al., 2004), and a seed collection plan for the
endangered and protected flora of Catalonia (oriented to capture up to 95% of their genetic
diversity; Membrives & del Hoyo, 2008), both actions sponsored by the Catalonia’s
autonomous government.
ACKNOWLEDGMENTS
We wish to especially thank the collaboration, both as data supplying as well as
suggestions, of the following researchers: Maria Renée Orellana, Ana Rovira and Julián
Molero (University of Barcelona), Gabriel Segarra-Moragues (Centro de Investigaciones
sobre Desertificación, CSIC), and José M. Iriondo (Universidad Rey Juan Carlos). The
present study has been carried out in the framework of the Research Grants REN2000-0829/
GLO, REN2003-01815 and CGL2007-60475/BOS (Ministerio de Educación y Ciencia), and
FBG 301022 (UB-Departament de Medi Ambient, Generalitat de Catalunya). Dr. Jordi
López-Pujol has benefited from two MEC fellowships (AP2000-1410 and EX2005-0922),
whereas Dr. Maria Bosch had a UB research contract of the “Ramón y Cajal” Program
(MEC).
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