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Author(s):
G. Bacchetta, G. Fenu, R. Gentili, E. Mattana and S. Sgorbati
Article title: Preliminary assessment of the genetic diversity in Lamyropsis microcephala (Asteraceae)
Article no:
TPLB_A_717548
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(XML)
Plant Biosystems, Vol. 00, No. 0, Month 2012, pp. 1–8
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Preliminary assessment of the genetic diversity in Lamyropsis
microcephala (Asteraceae)
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G. BACCHETTA1, G. FENU1, R. GENTILI2, E. MATTANA1, & S. SGORBATI2
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Centro Conservazione Biodiversita` (CCB), Dipartimento di Scienze della Vita e dell’Ambiente, Universita` degli Studi di
Cagliari, V.le Sant’Ignazio da Laconi 13, I-09123 Cagliari, Italy and 2Dipartimento di Scienze dell’Ambiente e del Territorio,
Universita` degli Studi di Milano-Bicocca, Piazza della Scienza 1, I-20126 Milano, Italy
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Abstract
Endangered species with small and isolated populations has been a key topic of conservation biology studies in the last
decade. Lamyropsis microcephala is among the most significant narrow endemic plants in the Mediterranean region, lying on
the Gennargentu massif of the Sardinia island (Italy). Due to heavy threat factors, this species has rapidly become threatened
with extinction. The inter-simple sequence repeat technique was used to assess the genetic variation and structure of the 80
individuals growing in the four remnant localities known to date, with the aim to implement further conservation strategies.
Results indicated a degree of differentiation among the four subpopulations, in particular for the Fonni one. The estimates of
Nei’s genetic diversity (H) ranged from 0.0563 (Fonni) and 0.1104 (Bau ‘e Laccos). Analysis of molecular variance values
showed that 53% of the total variation may be attributed to the individuals within subpopulations, while 47% is due to
differences among subpopulations (P 5 0.001). Results also highlighted a scarce gene flow (Nm ¼ 0.503).
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Keywords: Carduinae, inter-simple sequence repeats, narrow endemic, population genetics, Sardinia
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Introduction
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The theoretically founded aims of conservation
biology within a biogeographic context are the
preservation of members of a whole biota (individuals, populations, species, etc.) and the preservation of the functional ecological systems (Blondel
1999). Due to their evolutionary significance, the
endangered species with small and isolated populations have been key topics of conservation biology
studies in the last decade to understand the extinction mechanisms and avoid biodiversity loss (Beissinger 2000; Fre´ville et al. 2007; Abeli et al. 2009).
Lamyropsis (Charadze) Dittrich is a genus of the
Asteraceae family, member of the tribe Cardueae
Cass., subtribe Carduinae Dumort (Greuter &
Dittrich 1973), which comprises eight perennial
species distributed from southern Europe eastwards
to Southwest Asia (Mabberley 2008), most of which
are geographically isolated (Ha¨ffner 2000). This
genus, together with Carduus, Cirsium, Cynara,
Ptilostemon, Galactites, Notobasis and Tyrimnus con-
stitutes the Carduus–Cirsium clade, a geographically
homogeneous entity with a centre of distribution in
the Mediterranean area (Haffner & Hellwig 1999).
Lamyropsis microcephala (Moris) Dittrich & Greuter has been considered one of the most threatened
endemic of Sardinia (Arrigoni 1974), and it has been
included under the critically endangered (CR)
IUCN category in the national (Conti et al. 1992)
and regional (Conti et al. 1997) Italian Red Lists. In
2005, it has been listed among the ‘‘Top 50’’
threatened plants species of the Mediterranean
Islands (de Montmollin & Strahm 2005), in the
IUCN Red List under the CR category (Camarda
2006; Fenu et al. 2011) and in the European
threatened plant list (Bilz et al. 2011). A rapid
reduction of its distribution area, due to increasing of
tourist activities (ski area) and intense grazing, has
occurred in the last decades (Bacchetta et al. 2007;
Fenu et al. 2011). Such a reduction could lead to a
loss of genetic richness of the individuals of the
extant subpopulations, compromising species survival. Increasing habitat reduction and fragmentation
55
Correspondence: R. Gentili, Dipartimento di Scienze dell’Ambiente e del Territorio, Universita` degli Studi di Milano-Bicocca, Piazza della Scienza 1, I-20126
Milano, Italy. Tel: þ39 02 64482700. Fax: þ39 02 64482996. Email: [email protected]
ISSN 1126-3504 print/ISSN 1724-5575 online ª 2012 Societa` Botanica Italiana
http://dx.doi.org/10.1080/11263504.2012.717548
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G. Bacchetta et al.
may induce decline in the population size and
genetic diversity (Cole 2003; Matthies et al. 2004;
Leimu et al. 2006; Bruni et al. 2012), forcing wild
plant populations with reduced fitness to pass the
extinction threshold (Reed 2005).
The aim of this study was to assess the genetic
structure and the amount of genetic variation of L.
microcephala within and among subpopulations of the
extant four localities, and their gene flow, using
inter-simple sequence repeat (ISSR) molecular
markers.
Materials and methods
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Plant description
Lamyropsis microcephala, a perennial rhizomatous
herb, is a heliophile and mesophile species. The
flowering season starts at the end of June and finishes
in August, while the fruiting season begins at the end
of July, finishing in September (Bacchetta et al.
2007).
It grows on a glareicolous metamorphic substrata
in catchment areas and small valleys subjected to
long persistence of the snow and intense soil erosion.
The vegetation community where L. microcephala
grows is a perennial grassland, with hemicryptophytes and cushion chamaephytes being dominant,
characterized as the endemic Carici–Genistetea lobelioidis vegetation class. In the European Habitat
Directive (DIR 92/43/EEC), this vegetation type is
endemic oro-Mediterranean heaths with gorse (code
4090) and the subtype Cyrno-Sardinian hedgehogheaths (code 31.75).
The four known localities where L. microcephala
grows are situated in the northern slope of the Monte
Bruncu Spina in the municipality of Fonni and in the
western slope in the municipality of Desulo (Table
I). The average distance among localities ranges
from a minimum of 588 m between Fonni (Lf) and
Bruncu Spina (Ski) to a maximum of 2357 m,
between Desulo (Ld) and Bau ‘e Laccos (Baa), as
the crow flies.
175
Sampling operation
Plant material was collected from the four localities,
after obtaining the permits required by European and
national laws for the species listed in the appendices
of the Habitat Directive (DIR 92/43/EEC).
Young leaves of L. microcephala were collected in
2008 in the two historical localities from 20
individuals of the Ld, randomly chosen throughout
its area of distribution and from 9 individuals of the
Lf one. Two new localities were recently discovered
(Fenu et al. 2011); therefore, a new sampling was
carried out by collecting from 12 randomly chosen
individuals of the Ski and from 15 individuals of the
Baa (Table I). Reduced individual sampling (nine
individuals from the Lf), is due to the risk status of L.
microcephala in order to avoid any kind of impact on
the species as it grows in a very small surface area
(200 m2). According to Brainholt et al. (2009),
although sampling a high number of individuals per
population is optimal, in conservation genetic studies
rare species often have small sample sizes. This
sampling strategy is based on the precautionary
principle (Matsuda 2001) and has mostly been
applied to CR species (both plant and animal:
Brainholt et al. 2009; Ursenbacher et al. 2010), with
small–medium population size and to species with
clonal reproductive strategy, for which to assess the
exact number of genets and ramets can be difficult
(see Moreira et al. 2010; Smith & Waldren 2010).
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DNA isolation and PCR amplification
Genomic DNA was isolated from about 0.1 g of
frozen leave tissue using DNase plant mini kit
(Qiagen, Hilden, Germany), as specified by the 210
manufacturer. The DNA was suspended in 150 ml
of AE buffer (Qiagen).
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160
Table I. Number of sampled and total individuals of Fonni (Lf), Desulo (Ld), Bruncu Spina (Ski) and Bau ‘e Laccos (Baa) subpopulations
of L. microcephala.
Code
Sample size
Population
size (ramets)*
Population
area (m2)*
Fonni
Lf
9
2,066
200
Desulo
Ld
20
1,990,000
240,000
Bruncu Spina
Ski
12
5,196
600
Bau ‘e Laccos
Baa
15
116,875
12,500
Locality
165
170
Note: *Data from Fenu et al. (2011).
Central coordinates
(datum WGS84)
40801.3260
09818.2550
40801.0090
09817.7080
40801.1820
09818.3050
40800.8320
09819.5500
N
E
N
E
N
E
N
E
Elevation m
(a.s.l.)
1,580–1,590
220
1,450–1,820
1,625–1,637
1,450–1,590
225
Genetic diversity of L. microcephala
230
235
240
245
250
The PCR was carried out in a 25-ml total reaction
volume, including 30 ng total genomic DNA, 106
reaction buffer (Qiagen) and 10 mM of 17/18 bp
primers (Table II), 0.3 mM dNTPs in equal ratio and
AQ2 1 U of Taq DNA polymerase (Qiagen). The ISSR
primers used are reported in Table II. Top Taq
DNA polymerase was used at 1 U per reaction. The
ISSR-PCR amplification was performed in a Mastercycler Gradient thermal cycler (Eppendorf, Hamburg, Germany) under the following temperature
profile: 948C for 5 min to denature, 42 cycles at
948C for 40 s to denature, 558C for 40 s to anneal,
728C for 90 s to extend and finally 1 cycle at 728C
for 7 min.
The ISSR products were separated by electrophoresis in 2% MetaPhor agarose gel run with TBE
AQ3 buffer. Amplifications were performed at least twice
and only reproducible bands were taken into account
for further data analysis. Photographs (Gel Doc 2000
Biorad, USA) of the ethidiumbromide-stained gels
visualized by ultravoilet light illumination were
taken.
Data analysis
255
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270
Each amplified DNA band was scored using the
binary system that recorded the presence (1) or
absence (0) of the band. The binary matrix was
analysed using the software POPGENE version 1.32
(Yeh et al. 1997) assuming Hardy–Weinberg disequilibrium. Genetic diversity was estimated according to the following parameters: the percentage of
polymorphic bands (PPB%), Shannon’s index (Sh),
Nei’s gene diversity (H) and the gene differentiation
coefficient (GST) according to Nei (1973). The level
of gene flow (Nm) was measured following the
equation Nm ¼ (1/GST 7 1)/2 (Nei 1973).
To visualize genetic relationships among subpopulations, a dendrogram was constructed on the
Table II. ISSR primers, number of total reliable bands (NTB) and
NPB with PPB for each primer used in this study.
Primer
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280
285
UBC808
UBC811
UBC818
UBC822
UBC825
UBC826
UBC827
UBC830
UBC834
UBC862
Total
Mean
Sequence
(50 ! 30 )
(AG)8C
(GA)8C
(CA)8G
(TC)8A
(AC)8T
(AC)8C
(AC)8G
(TG)8G
(AG)8YT
(AGC)5
NTB
NPB
PPB%
11
13
15
12
10
9
6
9
12
7
104
10.4
7
9
12
10
4
4
3
9
8
1
67
6.7
63.64
69.23
80.00
83.33
40.00
44.44
50.00
100.00
66.67
14.29
64.42
3
basis of the unweighted pair-group method average
(UPGMA) and Nei’s genetic distance by using the
software POPGENE. Principal coordinates analysis
(PCoA) was performed to investigate and ordinate
associations between subpopulations with Dice
genetic distance matrix. Such analysis was carried
out by using the software PAST version 1.94
(Hammer et al. 2001).
Analysis of molecular variance (AMOVA) was
performed using Genalex software, version 6.1
(Peakall & Smouse 2006) to estimate the genetic
structure and degree of genetic differentiation within
and among the subpopulations of the four localities.
The test of significance for the AMOVA was carried
out on 999 data permutations.
To analyse the patterns of genetic variations along
bioecological gradients, Pearson’s correlation coefficient (r) was calculated, between Nei’s genetic
distance and the biotic/abiotic factors related to
the four localities. The following bioecological data
were obtained from Fenu et al. (2011): minimum,
mean and maximum value of altitude of the
localities, slope and aspect number of ramets,
percentage of reproductive ramets, number of
capitula per ramet and area (mq). The significance
of the genetic and geographic distances matrixes
was tested by the Mantel test, using 999 random
permutations. Both analyses were carried out with
the PAST program.
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Results
The 10 selected ISSR primers generated a total of
104 reproducible bands, with size ranging from 200
to 3000 base pairs across all samples of the four
subpopulations, with an average of 10.4 PCR
products per primer. The number of polymorphic
bands (NPB) was 67 (64.42%). The primers
UBC830 and UBC822 exhibited the highest level
of variability, yielding 100% and 83.33% of polymorphic bands, respectively (Table II).
At the subpopulation level, the PPB ranged from
14.42%, for both Lf and Ski, to 36.54% for the Baa
(Table III).
As for the genetic diversity distribution among the
four subpopulations, the estimates of Nei’s genetic
diversity (H) ranged from 0.0431 (Ski) and 0.11
(Baa; Table III). In addition, Shannon’s diversity
index showed a similar trend. Nei’s coefficient of
gene differentiation (GST) was 0.4984.
Results also highlighted a scarce gene flow
between the studied subpopulations (Nm ¼ 0. 5033,
Table III).
Nei’s distance index, based on UPGMA dendrogram (Figure 1(A)), revealed a differentiation of the
Lf from the others ones. The Ski and Baa are
grouped in a separate cluster.
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340
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G. Bacchetta et al.
Table III. Statistical analysis of genetic diversity in L. microcephala subpopulations (number in parentheses are standard deviation); NPB,
number of polymorphic bands; PPB%, percentage of polymorphic bands; na, observed number of alleles; ne, effective number of alleles; H,
Nei’s genetic diversity; Sh, Shannon index; GST, gene differentiation coefficient; Nm, gene flow.
Level
NPB
PPB%
Lf
Ld
Ski
Baa
Species
Mean
15
36
15
38
67
34.2
14.42
34.62
14.42
36.54
64.42
32.9
ne
na
1.1630
1.3804
1.1442
1.3654
1.6442
1.3288
(0.3714)
(0.4882)
(0.3530)
(0.4839)
(0.4811)
1.0983
1.1593
1.0692
1.1824
1.2285
1.1422
(0.2621)
(0.2898)
(0.1991)
(0.3085)
(0.3276)
H
0.0563
0.0982
0.0431
0.1104
0.1404
0.0866
(0.1420)
(0.1609)
(0.1179)
(0.1718)
(0.1749)
Sh
0.0840
0.1542
0.0666
0.1696
0.2233
0.1348
(0.2049)
(0.2354)
(0.1760)
(0.2507)
(0.2484)
GST
400
Nm
405
0.4984
0.5033
410
355
415
360
420
365
425
370
430
375
435
380
385
390
395
Figure 1. (A) The UPGMA dendrogram of the four subpopulations computed using the Nei’s genetic distance based on ISSR data. (B)
PCoA based on Dice genetic distances. The first two principal coordinates explained 34.32% and 16.13%, respectively, of the molecular
variance. The elliptic lines indicated 95% confidence intervals. Abbreviations: Lf, Fonni (square); Ld, Desulo (cross); Ski, Bruncu Spina
(dark grey circle); Baa, Bau ‘e Laccos (grey triangle).
In PCoA based on Dice distance between all pairs of
samples, the first two principal coordinates explained
34.32% (eigenvalue 0.195) and 16.13% (eigenvalue
0.092) of the molecular variance (Figure 1(B)).
The AMOVA values (Table IV), obtained by ISSR
data, showed that 53% of the total variation is
attributed to the individuals within while 47% is due
to differences among subpopulations (P 5 0.001).
Significant correlations according to Pearson’s
correlation (r) were found between Nei’s genetic
distance and two bioecological parameters: minimum value of altitude and slope (Table V(A)).
440
Mantel test revealed a non-significant correlation 445
between the genetic and geographical distance
matrixes (Table V(B); r2 ¼ 70.3553; P 4 0.05).
AQ4
Discussion
450
Reduced genetic diversity is often found in populations of endemic and rare plant species (Cole 2003;
Shao et al. 2009; Viana e Souza & Lovato 2010).
However, evidences that high genetic variability may
be retained by rare or narrowly distributed species 455
are also reported, especially in perennial species with
Genetic diversity of L. microcephala
Ellstrand and Roose (1987) reported that many
clonal species show a higher genetic variability than
their population size might suggest.
The L. microcephala subpopulations, ranging from
the two slopes of the Mount Bruncu Spina as a whole
(Bacchetta et al. 2007), were probably a unique
population in origin, then spatially fragmented
during the last century into four localities, distant
from 588 m to 2357 m (minimum distance), from
each other. This phenomenon was due to human
activities such as building of a ski run (1972–1973)
and overgrazing (Fenu et al. 2011). At present, these
four subpopulations could be considered only
partially isolated: (a) spatially as the large Ld is
located on the south-western Bruncu Spina slope,
while the other ones are situated on the north-eastern
slopes (Figure 1); (b) genetically, also due to the
mainly clonal reproductive characters of the species
(Diana Corrias 1977), that may favour reproductive
isolation also of closed localities, as detected by a low
level of gene flow.
In any case, the low Nm value could be due to
nature of ISSR makers that generally score a lower
rate of polymorphism with respect to other types of
markers (see Ci et al. 2008). We cannot exclude
residual genetic exchanges among subpopulations as
some species belonging to Carduinae subtribe close
to Lamyropsis (e.g. Cynara and Cirsium) are mostly
recent habitat fragmentation (Ge et al. 1997; Ayres &
Ryan 1999; Luan et al. 2006; De Vita et al. 2009;
Gentili et al. 2010).
In comparison with other species of the same
family, on the basis of ISSR markers, the present
study shows that L. microcephala conserves a moderate level of genetic diversity at the total population
level (PPB ranging from 14.42% to 36.54%; mean
32.9%; Table III). In particular, a PPB% comparable
to that present in L. microcephala was found in the
widespread Cirsium arvense populations (PPB ranging from 33% to 67%; mean 46.7%), belonging to
the same Cardueae tribe (Hettwer & Gerowitt 2004).
In Cirsium hillii, genetic differentiation among
populations was low, along with the presence of
private alleles in some populations and low gene flow
(Freeland et al. 2010). On the other hand, a work on
Carduus acanthoides (a ruderal species) revealed weak
evidence of inbreeding within populations and very
low genetic differentiation among populations (Mandak et al. 2009). Our data confirm previous papers
reporting that not all rare species, often present with
small populations, have a scarce level of genetic
diversity (Ranker 1994; Wang et al. 2006). In
particular, Mameli et al. (2008) detected a medium–high amount of genetic variability for another
remnant Sardinian endemic belonging to the Asteraceae family (Centaurea horrida Badaro`), while
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470
475
480
5
515
520
525
530
535
540
485
Table IV. The AMOVAs of the studied localities of L. microcephala based on 999 permutations. Abbreviations: df, degrees of freedom; SSD,
sum of square deviations; MSD, mean square deviations.
490
Source of variation
df
SSD
MSD
Estimated variance
Total variance (%)
P-Value
Among localities
Within localities
Total
3
52
55
180.183
235.626
415.808
60.061
4.531
4.081
4.531
8.612
47
53
100
50.001
50.001
545
550
495
AQ7
Table V. (A) Results of Pearson’s correlation between genetic diversity and 10 bioecological variables taken from Fenu et al. (2011). (B)
Results of Mantel’s test between the genetic (under the diagonal) and geographical (m; above the diagonal) distance matrixes.
A
500
505
555
Pearson’s r
P-Value
Density
No. of ramets
Reproductive
ramets (%)
No. of capitula
per ramets
Area
(mq)
Altitude
(min)
Altitude
(max)
Altitude
(mean)
Slope
Aspect
70.22
n.s.
0.38
n.s.
0.19
n.s.
0.72
n.s.
0.33
n.s.
70.96
0.04
0.22
n.s.
70.58
n.s.
70.97
0.03
70.39
n.s.
560
B
Code
510
Lf
Ld
Ski
Baa
Lf
Ld
Ski
Baa
–
0.0919
0.1767
0.171
588.11
–
0.1036
0.08
268.86
565.69
–
0.0241
2,042.34
2,357.07
1,862.91
–
Note: R ¼ 70.3553; P ¼ 0.876.
565
570
575
580
585
590
595
600
605
610
615
620
625
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G. Bacchetta et al.
pollinated by bees, as well as butterflies and beetles.
We hypothesized that such behaviour may also be
valid for L. microcephala. Nevertheless, even if bees
and other insects are able to fly for few kilometres
(Pasquet et al. 2009), while the majority of betweenflower flies occur within, very few occur between
even close plant localities. Moreover long-distance
pollen flow is very difficult to assess also by genetic
studies (Pasquet et al. 2009).
Although the genetic distances among subpopulations were low, our results showed a marked
distinction of the Lf one. In particular, according
to PCoA, we found a clear relationship between Ski
and Baa subpopulations but not between these and
the Lf one. This pattern suggests the presence of
recent genetic drift phenomena from the biggest Ld
subpopulation (in a central position with respect to
the others in the PCoA). The Ski and Baa likely have
a common origin or history, probably due to
extinction and colonization events and a recent
spatial fragmentation. Even if Lf is located no far
from the others ones, the scarce gene flow among
subpopulations (Nm ¼ 0.5033; Table III) and the
small size indicates a substantial isolation, probably
due to the disrupted interactions with pollinators
(Kearns et al. 1998; Jadwiszczak et al. 2011). On the
other hand, the small size of Lf and Ski makes them
extremely vulnerable to the effects of demographic,
environmental and genetic stochasticity (Menges
1998; Holsinger 2000; Fre´ville et al. 2007).
Despite the low number of subpopulations (determining a weak correlation), we found that
subpopulations with lower slope inclination and
reaching the bottom valleys (lower altitude) had
higher size and genetic diversity. This pattern could
be due to the following reasons: (a) growth sites with
lower inclination are more stable from a geomorphologic point of view, with a reduced rock/debris
fall activity and the presence of thicker soils with
higher surface cover; (b) lower inclination favours
persistence of snow cover, determining soil moisture
persistence until late summer and, therefore, favourable conditions for seed germination (Mattana et al.
2009); (c) more favourable growing conditions may
encourage a better reproductive biology. The Ld and
Baa subpopulations are likely to have a higher plant
viability in relation to suitability of habitat (Nicole`
et al. 2011), consequently higher individual number
and genetic diversity with respect to Lf and Ski.
However, endemic or narrow distributed species
with small populations may persist for long periods
in biogeographic niches and avoid extinction due to
their ecological specialization and clonal reproduction (Williams et al. 2009; Gentili et al. 2011).
In conclusion, although the limited sample sizes
(mainly for the Lf subpopulation), this study allowed
a preliminary assessment of this species in the
remnant localities where it lives to be carried out
and highlighted the importance of their protection
for the long-term conservation of this species. 630
However, a more exhaustive study should be carried
out in order to quantify the extinction risk of this
species and to plan correct conservation measures.
Acknowledgements
635
The authors wish to thank the Regione Autonoma
della Sardegna, for the financial support provided for
the activities carried out by the Centro Conservazione Biodiversita` (Project Sardegna 03, ‘‘Tutela di 640
specie vegetali endemiche esclusive della Sardegna
ad areale puntiforme ed a grave pericolo di estinzione’’).
The authors are grateful to Prof. Gianni Barcaccia
(Agripolis, University of Padova) for providing help- 645
ful critical comments on the manuscript.
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