Morphological, ecological and geneticvariability of Lavandula luisieri (Rozeira)Rivas-Martınez in central eastern Portugal

Fernanda Delgado1*, Sılvia Ribeiro2, Alvaro Alves1, Eliseu Bettencourt3

and Sonia Dias4

1Escola Superior Agraria do Instituto Politecnico de Castelo Branco, Quinta da Senhora de

Mercules, Apartado 119, 6000-909 Castelo Branco, Portugal, 2Departamento de

Proteccao de Plantas e de Fitoecologia, Instituto Superior Agronomia, Lisbon, Portugal,3Genetic Resources, Ecophysiology and Plant Breeding Unit, Instituto Nacional dos

Recursos Biologicos, I.P., Quinta do Marques, 2784-505 Oeiras, Portugal and 4Bioversity

International, Via dei Tre Denari, 472/a, 00057 Rome, Italy

Received 7 July 2009; Accepted 19 November 2009

AbstractThe morphological characterisation and data analysis of germplasm accessions of wild

Portuguese Lavandula luisieri (Rozeira) Rivas-Martınez from the southern Beira Interior

region of central eastern Portugal are described. The study, based on seeds and cultivated

plants, was conducted in 2005, 2006 and 2008 among populations selected from four sites

(I, II, III and IV). Quantitative and qualitative phenotypic data relating to a set of 35 morpho-

logical traits were analysed using canonical discriminant analysis. Genetic similarity among

accessions was assessed using amplified fragment length polymorphism molecular markers.

The traits contributing most to the variability among studied populations were related to

plant density, leaf colour, seed weight and various spike and flower characteristics. Plant

populations from each of the four test sites were statistically distinct, exhibiting unique

characteristics when compared with one another; however, populations from Sites II, III and

IV showed greater genetic similarity and differed substantially from the population of Site I.

Altitude and temperature were found to be the most significant environmental variables

influencing plant traits, yet the morphological variability of L. luisieri was also influenced

by soil pH levels, suggesting that the expressed variability is not only a result of genetic

characteristics but also of existing ecological conditions.

Keywords: ecology; genetic diversity; Lavandula luisieri; morphological traits; variability

Introduction

Lavandula luisieri (Rozeira) Rivas-Martınez (Rivas-

Martınez, 1979) [ ¼ L. stoechas L. subsp. luisieri (Rozeira)

(Rozeira, 1964) and ¼ L. stoechas subsp. linneana Rozeira

var. luisieri Rozeira (Rozeira, 1949)] is an aromatic plant

endemic to the southern region of the Iberian Peninsula

and is a part of the Stoechas section of the Lamiaceae

family. This plant is characteristic of the syntaxon

Cisto-Lavanduletae, a class which includes thermo- to

supra-Mediterranean dry and semi-arid, sub-humid second-

ary scrub communities producing aromatic compounds

(Rivas-Martınez et al., 2002a). L. luisieri exhibits ornamental,

melliferous and medicinal properties and serves as a natural

insect repellent (Eisner et al., 1986). Its essential oil contains

a series of compounds with the 1,2,2,3,4-pentamethyl-

cyclopentane (necrodane) structure, which also exhibits

insecticidal properties (Garcia-Vallejo et al., 1994; Delgado* Corresponding author. E-mail: fdelgado@esa.ipcb.pt

q NIAB 2010ISSN 1479-2621

Plant Genetic Resources: Characterization and Utilization 8(1); 82–90doi:10.1017/S1479262109990219

et al., 2006). Because of these qualities, L. luisieri may be

a viable species for alternative crop production in the

southern Beira Interior region of central eastern Portugal.

Conserving and cultivating the plant may also have

important implications for revitalising zones damaged by

the wild fires plaguing Portugal in recent decades.

The species L. stoechas displays a high morphological

variability, resulting in confusion over the correct taxo-

nomical identification of L. luisieri, as many species and

subspecies are commonly referred to as rosmaninho or

Portuguese Lavender. Further, the ssp. L. luisieri is part of

a distinct taxonomical section, Stoechas Ging. and exhibits

the chromosome number 2n ¼ 30 (Suarez-Cervera, 1986),

derived from the base numbers x ¼ 6 and x ¼ 9. This is

the only section in the Lavandula genera maintaining a

constant chromosome number, meaning the plant is an

amphidiploid (Garcia, 1942; Fernandes and Leitao, 1984)

and originates from the hybridisation of two species with

a diploid set of chromosomes from each parent. Like

other species in the Lavandula genera, L. luisieri can be

propagated from seed, by layering and cuttings. In the

wild, seed propagation is the dominant means of reproduc-

tion, enhancing the species’ variability. The flowering

period for L. luisieri generally occurs from February to

June (Upson and Andrews, 2004), but in central eastern

Portugal, flowering only takes place from March to June,

due to the dry, hot spring. The plant’s ripening period is

from June to July, and bees serve as the main pollinators.

In addition to the varietal characterisation of species,

which essentially relies on the use of molecular markers,

it is equally important to use morphological traits to

identify species present in the field and to serve as a

complementary tool to molecular biology, while also

considering phytosociological features. The main objec-

tive of this study was to identify and quantify the differ-

ences and similarities existing among selected L. luisieri

populations to determine the extent to which morpho-

logical, genetic and ecological factors are able to provide

parameters for species characterisation.

Materials and methods

Location selection and plant material

In 2005, wild seeds and whole plants selected from four

random populations of L. luisieri in a 50 m2 area of the

southern Beira Interior region were collected (Table 1).

Thirty plants from each of the four sites were maintained

as living collections in an experimental plot at ‘Quinta

da Senhora de Mercules – Escola Superior Agraria de

Castelo Branco’ (School of Agriculture of Castelo

Branco Polytechnic Institute), where the morphological

characterisation was conducted.

Table 1 shows the characterisation of the selected sites

based ongeographical, ecological and climatic parameters.

Floristic and environmental data recording

Field sampling was carried out in each of the four

L. luisieri sites (Table 1) based on the phytosociological

concepts of Braun-Blanquet (1979), modified by Gehu

and Rivas-Martınez (1981). L. luisieri communities were

surveyed using the Mueller-Dombois and Ellenberg (1974)

concept of minimum areas (the smallest areas adequately

representing community composition), and each percen-

tage of taxon cover was recorded by adapting the scale of

Braun-Blanquet (1964). The study adopted plant nomen-

clature based on Franco (1984), Franco and Rocha

Afonso (2003) and Castroviejo et al. (1993), Castroviejo

(1997). Syntaxa were named according to Rivas-Martınez

et al. (2001, 2002a, b).

A phytoecological characterisation was determined

for each collection site, including both qualitative and

quantitative environmental measurements: (1) bioclimatic

values defining thermotypes and ombrotypes (Rivas-

Martınez, 2005; Monteiro-Henriques, 2009); (2) edaphic

variables, including physiographic factors (altitude,

aspect and slope), geological factors (substrate rock

type) and pedological factors (texture, pH, potassium,

phosphorus and organic substrate).

Morphological data recording

Morphological observations were conducted on ten

living plants per population and were distributed in

three field replications using 35 morphological traits

(Table 2). The list of descriptors and methods of obser-

vation and data recording were consistent with those

developed by Bettencourt and Dias (2008). Plant colours

were compared and determined using the Royal Horticul-

tural Society (RHS) Colour Chart.

DNA extraction and amplified fragmentlength polymorphisms

Leaves from each of the four populations were collected

for DNA extraction after seed germination. Plant DNAzol

Reagent (Invitrogenw) was used to isolate and extract

three genomic DNA samples from each population.

Protocols supplied by Applied Biosystems were followed

to produce amplified fragment length polymorphisms

(AFLPs). Subsets of restriction fragments were amplified

using three pairs of selective primers (Eco þ ACA/

Mse þ CTA, Eco þ AGC/Mse þ CTT, Eco þ AAG/

MSE þ CAA) (Vos et al.,1995). Amplified fragments were

Morphological, ecological and genetic variability of Lavandula luisieri 83

separated using an ABI PRISMw 3100-Avant Genetic

Analyzer, and only those DNA sequences between 30 and

500 bp were recorded as present. A binary matrix was

produced for each selective primer pair combination used.

Statistical analysis of morphological andecological data

All the quantitative morphological data were analysed

according to the ANOVA methodology in order to

determine the population effect in ten plants (replications)

(Maxwell and Delaney, 2004). Analysis was conducted

using SPSS software (version 16.0) to select variables

with statistical significance in the order of P , 0.05.

Multiple discriminant analysis (Legendre and Legendre,

1998) was utilized to select morphological character-

istics providing higher discriminant power for the four

L. luisieri populations. Of the 35 morphological traits,

only quantitative descriptors, and those for which the

canonical discriminant functions were significantly

different, were used in the analyses.

The relationship between species occurrence and

environmental factors was assessed through canonical

correspondence analysis (CCA), using CANOCO 4.5 soft-

ware (ter Braak and Smilauer, 2002), while relationships

between ecological, morphological and genetic data

were assessed through principal component analysis

(PCA). All the data were standardized to improve normal-

ity, and a Monte Carlo permutation test (999 permutations)

was applied to detect and eliminate select correlated

CCA variables found to be redundant in comparison with

the ordination models, as well as to identify and select

key variables for testing (ter Braak and Smilauer, 2002).

Genetic data

Matrices outlining the presence and absence of poly-

morphisms between repetitions among the four sites

were generated using Microsoftw Office Excel. This was

done for each of the three combinations of primer pairs

used in DNA fragment amplification. Cluster analyses

were then performed and the Jaccard coefficient applied.

Data were further analysed using unweighted pair group

method with arithmetic mean clustering methodology,

and the final dendrogram was generated by Multivariate

Statistical Package software.

Results

Phytosociological and ecological analysis

The L. luisieri communities studied represent a sub-

serial stage of Holm Oak forests from the series PyroTable

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zing.

F. Delgado et al.84

bourgaeanae-Querco rotundifoliae S. [meso-Mediterranean,

dry to sub-humid, silicicolous of Luso-Extremadurense

Province (Lousa, 2004)] and Cork Oak forests of the series

Poterio agrimonioidis-Querco suberis S. [meso-Mediterranean,

sub-humid, silicicolous of Luso-Extremadurense Province

(Lousa, 2004)].

Floristic and ecological differences in and between

populations were identified through the CCA. Following

Table 2. Descriptors for morphological characterisation of Lavandula luisieri

Code Traits/descriptors Score code - descriptors state

1 Plant1.1 Growth habit 1-upright; 3-bushy; 5-globular; 7-prostrate1.2 Size 1-very small; 3-small; 5-medium; 7-large; 9-very large1.3 Density 1-loose; 3-medium; 5-dense

2 Leaf2.1 Intensity of green colour 1-light; 3-medium; 5-dark2.2 Intensity of grey tinge 1-absent or very weak; 3-weak; 5-medium; 7-strong; 9-very strong2.3 Colour 1-greyish; 3-green-greyish2.4 Leaf blade 1-entire; 3-weakly dissected; 5-strongly dissected; 7-crenate dissected;

9- pinnatifid; 11-sub pinnatifid2.5 Shape of leaf blade 1-linear; 3-linear-oblong; 5-lanceolate; 7-oblong; 9-oblong-lanceolate;

11-oblong-oblanceolate2.6 Margins of leaf blade 1-flat; 3-slightly revolute; 5-revolute; 7-strongly revolute2.7 Pilosity 1-glabrescent; 3-puberulent; 5-tomentose; 7-villose-pubescent

3 Flowering stem3.1 Length (mm)3.2 Diameter (mm)3.3 Length of the peduncle (mm)3.4 Section of the peduncle 1-quadrangular; 3-round3.5 Intensity of the green colour 1-very light; 3-light; 5-medium; 7-dark; 9-very dark3.6 Pubescence 1-weak; 3-medium; 5-strong

4 Spike4.1 Width (mm)4.2 Length (mm)4.3 Shape 1-narrow conical; 3-conical; 5-truncate conical; 7-cylindrical;

9-sub-cylindrical; 11-ovoid; 13-fusiform; 15-narrow trullate4.4 Section of the spike 1-quadrangular; 3-round4.5 Width of fertile bracts (mm)4.6 Shape of fertile bracts 1-linear; 3-linear-lanceolate; 5-Cuneiform; 7-obovate; 9-obovate-romboid;

11-romboid-ovate; 13-obovate-orbicular; 15-ovate-orbicular; 17-ovate-mucronate; 19-cordate-reniform; 21-cordate-mucronate; 23-obtriangular;25-sub-orbicular; 27-sub-rectangular; 29-cordate

4.7 Main colour of the fertile bracts 1-white; 3-green; 5-green-yellowish; 7-green-greyish; 9-violet; 11-purple-reddish; 13-brown; 15-purpule

4.8 Presence of bracteoles 1-sometimes present; 3-always present4.9 Infertile bracts 1-present; 3-absent4.10 Length of infertile bracts (mm)4.11 Shape of infertile bracts 1-linear; 3-elliptical; 5-oblong; 7-oblanceolate; 9-sub-orbicular; 11-ovate;

13-obovate; 15-obovate-orbicular; 17-spatulate4.12 Main colour of infertile bracts 1-white; 3-green; 5-greenish; 7-yellowish; 9-purple; 11-pink; 13-light

pink; 15-violet4.13 Undulation of margin of infertile bracts 1-weak; 3-medium; 5-strong

5 Flower5.1 Colour of calyx 1-greenish; 3-purple; 5-violet; 7-greyish5.2 Pubescence of calyx 1-tomentose; 3-puberulent; 5-villose; 7-scaly-villose; 9-hirsute5.3 Colour of corolla 1-white; 3-pink; 5-purple; 7-purple-blackish; 9-violet; 11-violet-whitish;

13-light- blue; 15-blue;17- dark-blue; 19- blue-purple5.4 Length of corolla (mm)5.5 Date of flowering (dd-mm-yyyy)

6 Seeds6.1 Weight of 1000 seeds (g)

Data were recorded on scale from 1 to 29 (Bettencourt and Dias, 2008).

Morphological, ecological and genetic variability of Lavandula luisieri 85

the Monte Carlo permutation test, altitude, phosphorus

and dry ombrotype were selected as the key environ-

mental variables for analysis as they best explained

the ordination model (Fig. 1). The total percentage of

accumulated variance among species was 78.9%.

Three of the four studied populations were determined

to be a sub-serial of Poterio agrimonioidis-Quercetum

suberis forest, while the remaining population

was identified as a sub-serial of Pyro bourgaeanae-

Quercetum rotundifoliae forest. The CCA ordination

diagram (Fig. 1) illustrates the existing altitudinal gradi-

ent between L. luisieri populations, isolating sites III

and IV from sites I and II. Axis 1 of the diagram separ-

ates the L. luisieri communities associated with Cytisus

striatus (Hill) Rothm. and Lavandula pedunculata

(Miller) Cav. subsp. sampaiana (Rozeira); these rep-

resent the populations of sites III and IV. The L. luisieri

communities from Site II, however, occur in dry

ombrotype and meso-Mediterranean thermotype and

are therefore associated to Retama sphaerocarpa

(L.) Boiss. that explains the observed difference in the

vegetation series.

Morphological data

According to the phenotypes of L. luisieri, plants from Site I

were characterized as small shrubs with bushy growth,

while those from sites II, III and IV exhibited a globular

growth habit. All the plants studied were of medium size

(,50 cm), with the colour of bushy plants varying bet-

ween greyish (RHS191A) and green-greyish (RHS189A).

In addition, all the plants exhibited a tomentose, linear

and revolute leaf blade with peduncles ranging from 37

to 45 mm. These trait parameters correspond to those

outlined by Franco (1984) and Upson and Andrews (2004).

Spikes varied from truncate-conical in sites I and II

to truncate-conical and cylindrical in sites III and IV. Spikes

determined to be 63–68mm in length by 12–17mm in

width were larger than those defined by the parameters

of Franco (1984), Tutin et al. (1981) and Upson and

Andrews (2004). Bracteoles were found to be both present

and absent, while infertile bracts were always present,

varying from 26 to 32mm, consistent with the parameters

of Franco (1984) and Upson and Andrews (2004).

The shape of infertile bracts was predominantly spathulate;

Fig. 1. Canonical correspondence analysis. Triplot showing sample populations (white circles), species types andenvironmental variables selected using Monte Carlo permutation tests. Codes are as follows: Adenocom, Adenocarpuscomplicatus; agrocast, Agrostis castellana; agrotrun, Agrostis truncatula; anarbell, Anarrhinum bellidifolium; andrinte,Andryala integrifolia; asphram, Asphodelus ramosus subsp. ramosus var. ramosus; brismaxi, Brisa maxima; callvulg,Calluna vulgaris; carlrace, Carlina racemosa; cistcris, Cistus crispus; cistlada, Cistus ladanifer; cistpsil, Cistus psilosepalus;crucangu, Crucianella angustifolia; cytistri, Cytisus striatus; dacthisp, Dactylis glomerata subsp. hispanica; daphgnid,Daphne gnidium; elaefoet, Elaeoselinum foetidum; ericarbo, Erica arborea; genitria, Genista triacanthus; haliocym,Halimium ocymoides; helistoe, Helichrysum stoechas; jasimont, Jasione montana; lavasamp, Lavandula pedunculata subsp.sampaiana; leonlong, Leontodon longirostris; philangu, Phillyrea angustifolia; puliodor, Pulicaria odora; pyrbour, Pyrusbourgaeana; querrot, Quercus rotundifolia; retaspha, Retama sphaerocephala; rosmoff, Rosmarinus officinalis; sangmino,Sanguisorba minor subsp. verrucosa; trifangu, Trifolium angustifolium; xolagutt, Xolantha guttata.

F. Delgado et al.86

however, in sites II and III, a select number were elliptical as

referred to in Upson and Andrews (2004); Franco (1984)

considers the shape to be oblanceolate. The main colour

of infertile bracts varied from light pink (RHS N81C) in Site

II to violet (RHS N81A) in sites I, III and IV. The calyx

was determined to be tomentose, and the colour of the

corolla was purple-blackish (RHS N186B); these parameters

are consistent with those of Franco (1984).

Of the 35morphological traits (9quantitative and25quali-

tative), those contributing most to the variability among

populations were related to plant density (medium to

dense); leaf colour (intensity of grey tinge; very weak to

weak); spike shape (cylindrical to ovoid); shape of fertile

bracts (ovate-mucronate to cordate-mucronate); main

colour of fertile bracts (green to violet); main colour of infer-

tile bracts [light pink (RHS 83D) to violet (RHS N81A)];

margin undulation of infertile bracts (weak to medium);

colour of calyx [greenish (RHS 143D) to violet (RHS 86B)];

length of corolla (6.0 to 6.2mm in sites I, III and IV; signifi-

cantly different than Site II with 4.05mm).

The canonical discriminant analysis (Fig. 2) illustrates

the genetic variation within the studied populations for

the first two functions: length of corolla (Function 1)

and length of peduncle, length of infertile bracts, length

of flowering steam and weight of 1000 seeds

(Function 2) and based on the eight parameters outlined

above. This accounts for 86.9% of the total variance

within the dataset. Function 1 displayed the highest per-

centage of variance among the four populations (60%),

while Function 2 represented 26.9% of the total variance

within dataset. These first two functions have a discrimi-

natory and significant value of Wilk’s Lambda.

Genetic analyses

The three AFLP primer combinations generated 550

fragments, of which 458 (83.4%) were polymorphic. The

highest number of polymorphic bands originated from

the primer combination Eco þ ACA/Mse þ CTA (174 poly-

morphisms in 207 fragments), and the highest percentage

(77%)of genetic variationwithin apopulationwasdetected

in the population of Site I. The percentage of genetic

variation within the populations at sites II and IV was

58.5 and 52.4%, respectively. The estimated Gst value was

0.525 (Hs ¼ 0.549 and Ht ¼ 0.261), indicating that both a

large amount of variation existed within populations and

that the most efficient primer combination for detecting

such variations was Eco þ AAG/Mse þ CAA (Gst ¼ 56.1).

Moreover, results revealed that in addition to displaying

genetic variation within individual site populations, genetic

differences also existed among the L. luisieri communities

from the four sites (Fig. 3). The greatest degree of genetic

similarity among populations was found between sites III

and IV, which exhibited 74% similarity. The population

from Site II was also genetically similar to those of sites III

and IV, with 66% similarity; however, the Site I population

was significantly different from the others, displaying a

similarity of only 48%. This reveals the weak geographical-

genetic distance relationship at sites II, III and IV and

the observed effect of habitat fragmentation at Site I.

Discussion

The L. luisieri population of Site I occurs in sub-humid

ombrotype and thermo-Mediterranean thermotype, in

areas with a larger bioclimatic differentiation than those

occupied by the other three populations. Site I included

Fig. 2. Canonical discriminant analysis. Biplot with thequantitative morphological traits. In this figure: VVR, Site I;M, Site II; CF, Site III and P, Site IV.

Fig. 3. The hierarchical dendrogram based on the Jaccardcoefficient showing the genetic relationships among thefour sampled populations of Lavandula luisieri in centraleastern Portugal.

Morphological, ecological and genetic variability of Lavandula luisieri 87

high-shrubs such as Pyrus bourgaeana Decne., Phillyrea

angustifolia L. and Erica arborea L., indicating a long

period of non-human disturbance [according to Rivas-

Martinez et al., (2002a, b) these species are characteristic

of Quercetea ilicis and Pistacio lentisci-Rhamnetalia

alaterni, related to the climatophylous vegetation and high

shrub communities]. On the other hand, in sites II, III and

IV, species associated with recent human disturbance,

such as Cistus crispus L. (Site II), Anarrhinum bellidifolium

(L.) Willd. (Site III) and Carlina racemosa L. (Site IV) were

identified. Figure 1 clearly illustrates this distinction.

Correlations between morphological and ecological

characteristics were analysed using CCA and the Monte

Carlo permutation test. The ecological variables of phos-

phorus and pH were selected for analysis, admitted with

P , 0.05, as they best explained the differences in mor-

phological traits. The percentage of accumulated variance

of species was 27.1%, of which 12.1% was explained

solely by varying pH levels, indicating that a small

portion of morphological variability is attributed to

environmental factors and the remaining mainly to

genetic factors. Figure 4 illustrates a PCA using all the

parameters (morphological, ecological and molecular),

with 100% of the cumulative percentage of variance for

the first two axes. This figure reveals that pH levels were

more strongly correlated to colour parameters (colour of

calyx and leaf intensity of grey tinge). The first PCA

axis separates studied sites according to a function of

altitude and temperature, identifying Site I as having

a higher degree of polymorphism due to increased

temperatures. Otherwise, Site I maintained the lowest

degradation degree in relation to the other sites.

The results from the genetic similarity test were sur-

prising. During the electropherograms readings, samples

from Site I generated results which could not be

replicated. A number of hypotheses were therefore

formulated to explain this phenomenon:

(1) A problem in field sampling existed: it is possible

that seeds from other species were inadvertently

harvested along with L. luisieri seeds, giving the

population a high heterogeneous genetic profile.

(2) A phenomenon of sympatric or parapatric specia-

tion: in this case, the population was geographically

isolated. The diversity of the ecological niche,

the ability to adapt to different habitats and natural

selection played a crucial role in the specific

differentiation of this population.

(3) A high degree of polymorphism in the genome of

individuals within the population is related to biodi-

versity, genetic variation and adaptation: these

results from evolutionary processes whereby traits

are inheritable but are modified by natural selection.

The AFLP method does not apply only to hardware

encoding and it may be that individuals have,

through evolutionary processes, generated a signi-

ficant rate of mutation in the population’s genetic

heritage. Since the non-coding genome was rarely,

if at all, impacted by natural selection forces, it is

likely that this brought a level of significant change

to the genetic heritage, as explained by the differ-

ences revealed in the PCA in Fig. 4. This population

exhibited phenotypic characteristics, such as the

undulation and main colour of infertile bracts, and

Fig. 4. Principal component analysis: biplot showing the morphological, environmental and genetic data. In this figure, Site I,1; Site II, 2; Site III, 3; and Site IV, 4.

F. Delgado et al.88

was unique from the other populations due to its

adaptation to the varying bioclimatic factors and

non-human disturbance. This hypothesis would

suggest that, due to the high level of genetic vari-

ability, the population at Site I was the most ancient.

Conclusions

This paper is the first to report morphological traits ana-

lyses and AFLP variation within four wild populations of

an endemic plant from the Iberian Peninsula, observed in

a one-year ex situ cultivation. It is believed that additional

questions about genetic diversity and variability within

populations will be generated with further analyses

using increased numbers of primer pairs. The results

presented by this study embody a remarkable feature:

the higher genetic variability of L. luisieri from Site I

suggests a strong correlation with non-human disturb-

ance. More than 50 years of habitat fragmentation and

non-mobilisation, factors identified in situ as the differ-

ences in bioclimatic conditions from the other test sites,

did not sufficiently explain the increased level of genetic

variability of the Site I population as compared with the

others. Therefore, preservation of populations in areas

with low human disturbance, through the establishment

of genetic reserves, would seem to be the most effective

measure for conserving the genetic variability of this

valuable, endemic Iberian species.

Acknowledgements

The authors wish to acknowledge Paula Palma and Isabel

Castanheira for their support in the development of data-

bases; Tiago Monteiro-Henriques for his contribution

on bioclimatic indices and Prof. Doutor Joao Pedro Luz

for the English revision. A portion of this study was

supported by the Leonardo da Vinci programme. The

morphological characterisation was carried out under

the research activities of the project, Agro 800 – ‘National

network for conservation and utilisation of aromatic and

medicinal plants’ at Escola Superior Agraria (School of

Agriculture of Castelo Branco Polytechnic Institute). The

ecological analysis was supported by the Foundation

for Science and Technology through the PhD project

SFRH/BD/29 515/2.

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