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Taxonomy, morphometric circumscription and karyology of the MediterraneanAfrican representatives of Ophrys sect. Pseudophrys (Orchidaceae)F. Amich a; M. García-Barriuso a; A. Crespí b; S. Bernardos a

a Evolution, Taxonomy and Conservation Group (ECOMED), Department of Botany, Faculty of Biology,University of Salamanca, Spain b Environment and Life Technological Studies Center (CETAV),Herbarium/Botanic Garden, University of Trás-os-Montes e Alto Douro, Portugal

Online Publication Date: 01 March 2009

To cite this Article Amich, F., García-Barriuso, M., Crespí, A. and Bernardos, S.(2009)'Taxonomy, morphometric circumscription andkaryology of the Mediterranean African representatives of Ophrys sect. Pseudophrys (Orchidaceae)',Plant Biosystems - AnInternational Journal Dealing with all Aspects of Plant Biology,143:1,47 — 61

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Taxonomy, morphometric circumscription and karyology of theMediterranean African representatives of Ophrys sect. Pseudophrys

(Orchidaceae)

F. AMICH1, M. GARCIA-BARRIUSO1, A. CRESPI2, & S. BERNARDOS1

1Evolution, Taxonomy and Conservation Group (ECOMED), Department of Botany, Faculty of Biology, University of

Salamanca, Spain and 2Environment and Life Technological Studies Center (CETAV), Herbarium/Botanic Garden,

University of Tras-os-Montes e Alto Douro, Portugal

AbstractOphrys sect. Pseudophrys shows a pronounced morphological variation, especially in Mediterranean Africa, a centre ofradiation of this section. In Tunisia, different authors recognize between 9 and 13 species (and 5 subspecies). A multivariateanalysis was performed in order to study the polymorphisms of eight critical species of Ophrys sect. Pseudophrys representedin Tunisia (O. africana, O. aspea, O. battandieri, O. eleonorae, O. gazella, O. numida, O. subfusca and O. vallesiana). Thevariation in 20 floral characters (including 5 ratios) were evaluated in 332 live plants belonging to 20 populations ofthe mentioned Tunisian representatives of sect. Pseudophrys. Two additional labellum characters were measured for thepopulations belonging to O. subfusca group (namely O. aspea, O. battandieri, O. numida and O. subfusca). In order todistinguish the different species, and identify characters defining their circumscription, we carried out detailedmorphological analyses on two different data sets of sect. Pseudophrys, one consisting of all the currently recognizedspecies, and another consisting of all the populations belonging to the controversial O. subfusca group. In order to summarizethe overall morphological variation among Pseudophrys, a principal components analysis (PCA) and a cluster analysis ofpopulations were conducted. To test the separation of population groups resulting from the PCA and cluster analyses,a canonical discriminant analysis (CDA) was conducted. The most discriminating characters are presented in the form ofboxplots. Due to extensive overlapping in even the most distinguishing characters, it is not possible to accept O. africana as adistinct species from O. gazella, or O. numida as distinct from O. subfusca. Cytological data and chromosome counts arereported for the first time for eight Tunisian members of sect. Pseudophrys. All of the analysed species are diploids with2n¼ 36 chromosomes. Neither polyploids nor aneuploids were detected. A key to the accepted species is provided.

Keywords: Chromosome counts, Mediterranean Africa, multivariate morphometrics, Ophrys, Pseudophrys, taxonomy,Tunisia

Introduction

The genus Ophrys L. illustrates in a particularly

evident way the phenomenon of radiation by floral

differentiation, with bottlenecks, founder effects and

abrupt cladogenesis essential to the diversification

of the Orchidaceae (Benzing 1987; Devillers &

Devillers-Terschuren 2000b). Mediterranean Africa

appears to have been a centre of radiation for Ophrys

sect. Pseudophrys. It is also the only region where

sect. Pseudophrys generally exceeds sect. Ophrys in

frequency, abundance and diversity (Devillers &

Devillers-Terschuren 2000a). The pronounced

morphological diversity in this area is particularly

striking in the O. fusca complex, and is expressed by a

great microdiversity of floral details characterizing

relatively homogeneous swarms, separated or not in

space or time. The overall complex is characterized

by considerable similarity, even in floral characters.

Much of this microdiversity reflects adaptations to

different pollinators, as first described by Godfery

(1930) and later examined in detail by Paulus and

Gack (1981, 1995, 1999).

The number of species formally defined and

described under sect. Pseudophrys has tripled during

the last 10 years (Delforge 2002a, 2005). However,

Correspondence: Sonia Bernardos, Evolution, Taxonomy and Conservation Group (ECOMED), Department of Botany, Faculty of Biology, University of

Salamanca, 37008 Salamanca, Spain. Tel: þ34 923 294469. Fax: þ34 923 294484. Email: [email protected]

Plant Biosystems, Vol. 143, No. 1, March 2009, pp. 47–61

ISSN 1126-3504 print/ISSN 1724-5575 online ª 2009 Societa Botanica Italiana

DOI: 10.1080/11263500802633485

Downloaded By: [Dr, Francisco Amich] At: 15:48 10 June 2009

few cytological or molecular studies have been

undertaken, so the genetic distinction and phyloge-

netic relationships of the many proposed taxa remain

unclear.

Among recent estimates of the taxonomic diversity

of the sect. Pseudophrys in Tunisia, the most

conservative is that of Strohle (2003), who suggests

that nine species and five subspecies should be

Table I. List of populations and number of plants (N) of sect. Pseudophrys used for morphometric analyses, chromosome number (2n), and

voucher number in SALA. Abbreviations: TU, Tunisia. Collectors: DT, D. Tyteca; FA, F. Amich; SB, S. Bernardos. NKS, number of

karyological sample.

Taxon/Number and code of populations/Origin and collection data N 2n NKS Voucher

Ophrys africana G. Foelsche & W. Foelsche

OA01 TU SALA

Cap Bon, El Haouaria, 43 m, 32SPF7094 (36859010N–108540590 0E), 18.3.2005, FA & SB 16 – – 110686

Ophrys aspea Devillers-Tersch. & Devillers

OP02 TU SALA

Cap Bon, Korbous, Djebel Korbous, 173 m, 32SPF4077 (36850070 0N–108340330 0E),

18.3.2005, FA & SB

19 36 OP25/05 110641

Ophrys battandieri E.G. Camus

OB03 TU SALA

Bizerte, Utique, 155 m, 32SNF9099 (378020020 0N–108010160 0E), 27.3.2005, FA & SB 25 36 OB70/05 110718

OB04 TU SALA

Bizerte, Utique, 160 m, 32SNF9199 (378020030 0N–108010160 0E), 27.3.2005, FA & SB 18 – – 110719

TU SLIDE

Cap Bon, NE El Haouaria, 195 m, 32SPF8003, 4.3.2002, SB & DT – 36 OB21/02 10053

Ophrys eleanorae Devillers-Tersch. & Devillers

OE05 TU SALA

Sidi Thabet, Djebbes II, 275 m, 32SNF9581 (368520190 0N–108040030 0E), 17.3.2005, FA & SB 17 36 OE02/05 110590

OE06 TU SALA

Sidi Thabet, Djebbes II, Jebel Ahmar, 224 m, 32SNF9781 (368520390 0N–108050260 0E),

17.3.2005, FA & SB

15 36 OE04/05 110663

OE07 TU SALA

Sidi Thabet, Djebbes I, 195 m, 32SNF9582 (368520460 0N–108040370 0E), 17.3.2005, FA & SB 23 – – 110722

OE08 TU SALA

Teboursouk, road to Thibar, 560 m, 32SNF1737 (368280410 0N–98110380 0E), 26.3.2005, FA & SB 6 – – 110723

Ophrys gazella Devillers-Tersch. & Devillers

OG09 TU SALA

Sidi Thabet, Djebbes II, 275 m, 32SNF9581 (368520190 0N–108040030 0E), 17.3.2005, FA & SB 20 – – 110687

OG10 TU SALA


17.3.2005, FA & SB

18 36 OG10/05 110682

OG11 TU SALA


OG12 TU SALA

Teboursouk, near Dougga ruins, 625 m, 32SNF1832 (368260170 0N-98120220 0E), 26.3.2005, FA & SB 16 36 OG32/05 110688

Ophrys numida Devillers-Tersch. & Devillers

ON13 TU SALA

Teboursouk, near Dougga ruins, 625 m, 32SNF1832 (368260170 0N–98120220 0E), 26.3.2005, FA & SB 23 36 ON44/05 110640

Ophrys subfusca (Reichb. f.) Hausskn.

OS14 TU SALA


17.3.2005, FA & SB

22 36 OS15/05 110649

OS15 TU SALA

Teboursouk, road to Thibar, 560 m, 32SNF1737 (368280410 0N–98110380 0E), 26.3.2005, FA & SB 21 36 OS52/05 110635

OS16 TU SALA


Ophrys vallesiana Devillers-Tersch. & Devillers

OV17 TU 14 SALA

Sidi Thabet, Djebbes II, 275 m, 32SNF9581 (368520190 0N–108040030 0E), 17.3.2005, FA & SB 14 – – 110630

OV18 TU SALA

Sidi Thabet, Djebbes I, 195 m, 32SNF9582 (368520460 0N–108040370 0E), 17.3.2005, FA & SB 7 36 OV22/05 110597

OV19 TU SALA


OV20 TU SALA

Teboursouk, near Dougga ruins, 625 m, 32SNF1832 (368260170 0N–9812220 0E), 26.3.2005, FA & SB 14 36 OV65/05 110720

48 F. Amich et al.


recognized. At the other extreme, Delforge (2005)

recognizes 13 species, half of which have been

described in recent years (Delforge et al. 1991;

Devillers & Devillers-Terschuren 1994, 2000a,

2000b; Foelsche & Foelsche 2001). These taxa are

morphologically difficult to distinguish from one

another because of the lability and inconsistency of

the traits used in their identification, including the

variation in size, pilosity and colour of the labellum.

Highly contrasting and diverse taxonomic interpreta-

tions are therefore common (e.g. Baumann &

Kunkele 1986; Paulus & Gack 1995, 1999; Devillers

& Devillers-Terschuren 2000c; Golz & Reinhard

2000; Foelsche & Foelsche 2001, 2004; Delforge

2002b; Strohle 2003). A significant number of

nomenclatural problems remain unsolved, despite

recent advances in this area (e.g. Delforge 1999,

2004a, 2004b; Devillers & Devillers-Terschuren

2000c). Few attempts at circumscribing and ranking

taxa under sect. Pseudophrys based on multivariate

analysis of morphometric and/or molecular data have

been made to date (e.g. Golz & Reinhard 1990;

Lowe et al. 2001; Soliva et al. 2001; Bateman et al.

2003; Bernardos et al. 2005, 2006).

Here we present a detailed morphometric and

karyological analysis of Tunisan representatives of sect.

Pseudophrys. The main objectives were (1) to examine

the morphological and karyological variation within and

between Tunisian populations, (2) to clarify the

taxonomic positions of Tunisian species assigned to

the critical O. subfusca group (O. aspea, O. battandieri,

O. numida and O. subfusca), and (3) to evaluate the traits

that have been used to define taxa, and to establish a

comprehensive taxonomic treatment.

Material and methods

Plant material

In February–March of 2002, a field trip to Tunisia

was undertaken to locate, study and photograph

different populations of taxa belonging to sect.

Pseudophrys. In March–April 2005, a second field

trip was undertaken to collect material and to study

these populations morphometrically. A Garmin

e-map GPS was used to geographically locate the

populations using 161 km UTM and geographic

coordinates. A representative voucher specimen from

each population was collected and deposited at SALA

(Herbarium of the University of Salamanca, Spain).

Twenty Tunisian populations belonging to eight

putative species of sect. Pseudophrys were sampled for

morphometric analyses (Table I, Figure 1). Sixteen

to 25 plants per population sample (332 individuals

in total) were used for morphometric analyses; as

some populations were small, fewer individuals were

Figure 1. Map showing the distribution of the sampled populations of sect. Pseudophrys in Tunisia. Scale bar¼150 km.

Ophrys sect. Pseudophrys 49


sampled (6–12 plants). One population, from the type

locality (locus classicus), was sampled for Ophrys

africana G. Foelsche & W. Foelsche; one population

from the type locality, for O. aspea Devillers-Tersch.

& Devillers; two populations for O. battandieri E.G.

Camus; four populations for O. eleonorae Devillers-

Tersch. & Devillers; four for O. gazella Devillers-

Tersch. & Devillers; one, out of the only two

populations known in Tunisia, for O. numida

Devillers-Tersch. & Devillers; three for O. subfusca

(Reichb. f.) Hausskn.; and four (including the locus

classicus of the species) for O. vallesiana Devillers-

Tersch. & Devillers.

Twenty floral characters (including five ratios)

were measured. Two additional labellum characters

were measured for the populations belonging to

O. subfusca group.

Chromosome numbers

In this study, we examined 12 accessions, repre-

senting 7 species of Ophrys sect. Pseudophrys.

Karyological observations were based on material

collected from natural populations from various

localities in the range of the species. The chromo-

some number of two to four plants per population

was checked. For O. aspea and O. numida, we only

examined one population because of the scarcity and

restricted distribution of these taxa. For O. africana

we also examined one population. Unfortunately, the

quality and clarity of the plates did not allow

us to obtain any result. For the remaining five

species we karyologically analysed at least two

populations.

Voucher specimens or photographs are deposited

at SALA. Table I shows the origin of the samples.

For two species (O. aspea and O. vallesiana) we

collected the material in type localities. In Table II

we present some previous counts of central–western

Mediterranean Pseudophrys.

Chromosome counts were made using ovaries

sampled at a very early stage of development. Young

flower buds were fixed in absolute ethanol–glacial

acetic acid (3:1, sometimes modified to 6:1). Fixed

material was stored at 48C until staining with 2%

acetic orcein. Mounting involved squashing in 45%

acetic acid. At least three counts were made for

each population. A Nikon eclipse 50i microscope

connected to a Nikon Coolpix 5400 digital camera

was used to take photomicrographs. Drawings and

photomicrographs are deposited at the Department

of Botany of the University of Salamanca.

Analyses of morphological traits

The characters selected (Table III, Figure 2; see also

Bernardos et al. 2005, figure 2) include variables of

continuous variation (characters 1 – 15). In addition,

five ratios were computed (Table III: characters 16–

20), and two labellum characters (characters 21–22,

Table III, Figure 2) were specifically selected for the

O. subfusca group (namely O. aspea, O. battandieri,

O. numida and O. subfusca). Additionally, the

convexity of the labellum at the base and at the apex

was scored, but not used in multivariate analyses as

these characters did not vary within populations and

taxa. The data matrix used in this study is available

on request from S.B. ([email protected]).

Morphological characters were scored from plants

of natural populations. Floral parts from the most

recently opened flower on the stem, carefully checking

that it was always well-shaped and that there were no

significant differences in relation to the lowest flowers

on the stem, were measured in the field using an

electronic digital ruler. The traits examined were

selected according to those reported in previous

studies on sect. Pseudophrys (Golz & Reinhard 1990;

Arnold 1999; Lowe et al. 2001; Bernardos et al. 2005)

and our initial field observations.

Multivariate analyses were performed on: (1) the

whole Tunisian material of section Pseudophrys, in

order to resolve the overall patterns of variation and

to uncover morphological discontinuities among the

taxa studied, and (2) samples of the O. subfusca

group, to evaluate the variation between populations

and putative species.

To summarize the overall patterns of morphologi-

cal variation, a PCA (R type based on a correlation

matrix; Sneath & Sokal 1973; Krzanowski 1988)

and a cluster analysis (Everitt 1993; UPGMA:

unweighted pair-group method using arithmetic

Table II. Previous counts in species of the section Pseudophrys in

the central–western Mediterranean Basin.

Species Authors 2n Country

Ophrys casiella D’Emerico et al. (2005) 36 Sicily

Ophrys dyris Bernardos et al. (2003) 72, 90 Spain

Ophrys fusca Greilhuber and

Ehrendorfer (1975)

72 Balearic

Islands

D’Emerico et al. (2005) 36 Italy,

Sardinia

Bernardos et al.

(unpub. data)

72 Spain

Ophrys iricolor Scrugli (1977) 36 Italy

D’Emerico et al. (2005) 36 Sardinia

Ophrys lupercalis D’Emerico et al. (2005) 36 Sicily

Ophrys lutea Love and Kjellqvist

(1973)

36 Spain

Bernardos et al. (2003) 36 Spain

D’Emerico et al. (2005) 36 Italy

Ophrys sicula D’Emerico et al. (2005) 36 Italy,

Sardinia,

Sicily

Ophrys sulcata Balayer (1986) 72 France

Ophrys vasconica Bernardos et al. (2003) 72, 74 Spain

50 F. Amich et al.


averages to test for hierarchical structure) were

performed. Populations were represented by the

mean values of the traits studied. Prior to the

clustering analysis all data were standardized by

zero mean and unit standard deviation. City-block

(Manhattan) distance was used for calculating

pairwise similarities between OTUs.

To test the separation of population groups

delimited according to the PCA and cluster analysis,

canonical discriminant analyses (CDA; Klecka 1980;

Krzanowski 1988) were performed. Population

groups, populations and individual plants were used

as OTUs in these analyses. The total canonical

structure, expressing the correlation between mor-

phological traits with the canonical axes, was calcu-

lated to identify the characteristics responsible for

separating the groups. The CDA is considerably

robust for data sets deviating from multivariate

normality and equality of within-group covariance

matrices (Sneath & Sokal 1973; Thorpe 1976; Klecka

1980). Multivariate normality is not required when

CDA is used as an ordination procedure, and no

further statistical tests are necessary (Pimentel 1981).

Descriptive statistical values (means, medians, stan-

dard deviations and percentiles) were calculated for all

quantitative traits for all populations. The most dis-

criminating traits are presented in the form of boxplots,

with boxes defined as interquartile ranges and with the

whiskers representing the 10% and 90% ranges.

Morphometric analyses were done by using the

SPSS version 11.5 (SPSS 2002) and STATISTICA

version 7.0 (StatSoft 2004) software.

Results

Chromosome numbers

We report here, for the first time, the somatic

number of the seven Pseudophrys species studied. In

Tunisian Pseudophrys we have only detected one

Figure 2. Some quantitative morphological characters examined in

this study: WPB, DMS, MCL and WYM (see Table III). Scale

bar¼5 mm. A, Ophrys vallesiana; B, O. gazella; C, O. aspea; D, O.

battandieri; E, O. numida; F, O. subfusca.

Table III. List of characters measured and scored for morpho-

metric analyses of Ophrys sect. Pseudophrys in Tunisia.

Morphological character Initials

Character

states

1 Length of sepals LS mm

2 Width of sepals WS mm

3 Length of petals LP mm

4 Width of petals WP mm

5 Length of labellum LL mm

6 Width of labellum WL mm

7 Length of lateral

lobe of labellum

LLL mm

8 Width of lateral

lobe of labellum

WLL mm

9 Width of central

lobe of labellum

WCL mm

10 Length of speculum LSP mm

11 Distance base

speculum–throat

SPT mm

12 Distance base

speculum–sinus

SPS mm

13 Width of the labellum

at the base

WLB mm

14 Width of the plateau

at the base of labellum

WPB mm

15 Width of yellowish margin WYM mm

16 Ratio length/width of labellum LL/WL

17 Ratio length of labellum/

length of lateral lobe

LL/LLL

18 Ratio length of labellum/

length of speculum

LL/LSP

19 Ratio width of labellum/

length of speculum

WL/LSP

20 Ratio width of labellum in the

base/width of plateau

WLB/WPB

21 Width of yellowish margin

up to central lobe

MCL mm

22 Width of yellowish margin

up to sinus

DMS mm



ploidy level: diploid (2n¼ 36). No aneuploid phe-

nomena were observed.

Analyses of morphological traits

All Tunisian representatives of sect. Pseudophrys.

Table IV shows the basic statistical parameters for all

characters examined in each taxon.

The cluster analysis based on population values

divided these populations into three main clusters

(Figure 3). The three clusters correspond to the

three traditionally recognized groups of taxa (cluster

1: O. eleonorae þ O. vallesiana; cluster 2: O. africana

þ O. gazella; cluster 3: O. subfusca group). The PCA

diagram based on all populations (Figure 4) shows

three groupings of populations corresponding to the

same three groups of species. In the plot based on

population values (Figure 4), the O. subfusca group is

clearly separated along the first axis (representing

64.85% of the total variation), whereas the group

consisting of O. eleonorae and O. vallesiana is

separated from O. africana and O. gazella along the

second axis (representing 23.82% of the total

variation). Thirteen of the 20 characters contributed

almost equally to separation along the first axis, and

7 characters contributed to separation of the groups

along the second axis (Table V A).

In the ordination diagram of the CDA (figure not

shown), populations of all three groups were clearly

separated along the two canonical axes. The first

canonical axis (representing 91.89% of the variation)

is mainly correlated with character WYM and ratios

WLB/WPB and LL/LLL (Table V B). The main

characters correlated with the second canonical axis

(representing 7.19% of the variation) are LLL and

SPS (Table V B).

The most discriminating characters are presented

as boxplots (Figure 5).

Tunisian representatives of the O. subfusca group

(namely O. aspea, O. battandieri, O. numida and O.

subfusca). The cluster analysis based on population

values divided the population into two main clusters

(Figure 6), one corresponding to O. battandieri and

the other encompassing the remaining taxa.

The PCA plot based on population values

(Figure 7) showed two groups of populations and

two isolated populations. The only population of O.

aspea was clearly separated along the first axis

(representing 66.82% of the total variation). The

remaining populations were separated along the

second and the third axis (representing 12.80% and

7.45% of the total variation, respectively). More than

half of the characters (12 of 22) contributed almost

equally to separation along the first axis, whereas 11

and 10 characters contributed to separation of the

groups along the second and third axis, respectively

(Table VI A).

In the ordination diagram of the CDA (Figure 8),

individual populations of O. aspea and O. battandieri

are nearly consistently separated along the first axis

(representing 79.14% of the total variation); the

remaining variation is broadly overlapping between

populations and taxa (Figure 8). Several floral

characters (i.e. WYM and DMS) contributed mostly

to separation of OTUs along the first axis, whereas

only the SPT, and ratios LL/LSP and WL/LSP were

more important for separation along the second axis

(Table VI B).

A univariate examination of selected morphologi-

cal characters among populations of the O. subfusca

group showed that the yellowish margin up to the

sinus (DMS) is consistently absent in O. aspea but

clearly obvious in the remaining three species

(Figure 9B). The width of the yellowish margin up

to the central lobe (MCL, Figure 9A) is a highly

variable character in O. battandieri, and a scarcely

variable one in O. aspea.

Discussion and conclusions

Chromosome numbers

Chromosome numbers for O. aspea, O. battandieri,

O. eleonorae, O. gazella, O. numida, O. subfusca and

O. vallesiana are reported for the first time in the

present contribution (Figures 10 and 11). Previous

karyological studies have shown the basic haploid

chromosome number of the genus Ophrys to be

x¼ 18 (e.g. Greilhuber & Ehrendorfer 1975). How-

ever, tetraploid taxa with 2n¼ 4x¼ 72 have

been encountered, as well as pentaploids with

2n¼ 5x¼ 90 (Greilhuber & Ehrendorfer 1975;

Bernardos et al. 2003).

Polyploidy has only been detected in members of

sect. Pseudophrys (i.e. O. dyris, O. fusca and O.

vasconica) and only in material from the Iberian

Peninsula and the Balearic Islands. A recent study of

the karyomorphology of Ophrys (D’Emerico et al.

2005) reported all the Pseudophrys taxa studied (from

mainland Italy, Sardinia and Sicily) to be diploid

with 2n¼ 2x¼ 36 (see Table II).

The seven species analysed in the present work were

also diploid (2n¼ 2x¼ 36). Thus, to date, polyploid

taxa have only been found in the Iberian Peninsula

while only diploids have been found in other parts of

the Mediterranean Basin. No cases of somatic

aneuploidy were seen in the present study, a phenom-

enon reported in other cytological studies (e.g.

Greilhuber & Ehrendorfer 1975; Bianco et al. 1991).

Of the 67 species recognized by Delforge (2005) to

be members of sect. Pseudophrys, karyological data

are available for only about 10% (for material mainly

from the central–western Mediterranean) (Table II).

A karyological study over a broader distribution

52 F. Amich et al.


Table IV. Summary statistics for 15 characters examined in Ophrys africana (OA), O. aspea (OP), O. battandieri (OB), O. eleonorae (OE), O.

gazella (OG), O. numida (ON), O. subfusca (OS) and O. vallesiana (OV). Characters MCL and DMS were examined in the O. subfusca group

only.

Character Taxa Mean Median

Standard

deviation

10–90

percentile Character Taxa Mean Median

Standard

deviation

10–90

percentile

LS OA 9.10 9.17 0.50 8.35 – 9.70 LSP OA 7.25 7.13 0.57 6.64 – 8.14

OP 9.16 9.17 0.77 8.20 – 10.15 OP 4.91 5.15 1.15 3.47 – 6.34

OB 10.10 10.10 0.78 9.07 – 11.19 OB 6.91 6.99 1.11 5.41 – 8.27

OE 13.82 13.83 1.28 12.38 – 15.32 OE 10.71 10.78 1.16 9.01 – 12.06

OG 9.28 9.34 0.88 8.27 – 10.42 OG 6.82 6.79 1.01 5.19 – 8.03

ON 9.28 9.25 0.86 8.47 – 10.56 ON 4.28 4.20 0.66 3.31 – 5.19

OS 9.64 9.45 1.16 8.35 – 11.70 OS 4.72 4.66 0.84 3.49 – 6.03

OV 11.97 12.00 0.97 10.66 – 13.04 OV 9.20 9.09 1.10 7.96 – 10.47

WS OA 5.18 5.20 0.55 4.51 – 5.83 SPT OA 0.43 0.00 0.73 0.00 – 1.58

OP 5.47 5.60 0.52 4.68 – 6.09 OP 1.81 1.97 0.67 0.92 – 2.58

OB 6.37 6.36 0.63 5.65 – 7.17 OB 0.92 0.00 1.01 0.00 – 2.26

OE 7.69 7.56 0.77 6.76 – 8.90 OE 0.47 0.00 0.80 0.00 – 1.68

OG 5.20 5.15 0.60 4.53 – 6.09 OG 0.20 0.00 0.50 0.00 – 1.27

ON 4.93 4.90 0.48 4.37 – 5.35 ON 1.37 1.44 0.83 0.00 – 2.36

OS 5.52 5.45 0.57 4.89 – 6.42 OS 1.70 1.81 0.64 1.06 – 2.42

OV 6.51 6.43 0.64 5.63 – 7.34 OV 0.49 0.00 0.80 0.00 – 1.71

LP OA 5.80 5.69 0.50 5.11 – 6.50 SPS OA 1.15 1.16 0.63 0.37 – 2.08

OP 5.78 5.95 0.60 5.16 – 6.35 OP 1.42 1.36 0.42 0.98 – 2.09

OB 5.93 5.90 0.55 5.24 – 6.53 OB 2.16 2.15 0.51 1.52 – 2.72

OE 8.72 8.65 0.77 7.92 – 973 OE 0.93 0.83 0.55 0.00 – 1.66

OG 5.79 5.69 0.73 5.02 – 6.70 OG 0.82 0.77 0.54 0.00 – 1.56

ON 5.39 5.21 0.52 4.93 – 6.20 ON 2.48 2.47 0.46 1.91 – 3.10

OS 6.07 5.85 0.98 5.15 – 7.94 OS 2.58 2.52 0.60 2.01 – 3.49

OV 7.61 7.73 0.64 6.79 – 8.33 OV 0.68 0.70 0.56 0.00 – 1.41

WP OA 2.04 2.04 0.25 1.76 – 2.37 WLB OA 4.19 3.96 0.67 3.75 – 5.02

OP 1.93 1.87 0.30 1.58 – 2.46 OP 3.77 3.78 0.34 3.21 – 4.22

OB 2.50 2.48 0.30 2.14 – 2.81 OB 4.82 4.78 0.45 4.37 – 5.48

OE 2.89 2.90 0.36 2.43 – 3.29 OE 4.89 4.83 0.55 4.23 – 5.69

OG 2.06 2.05 0.34 1.60 – 2.43 OG 4.00 3.99 0.51 3.42 – 4.52

ON 1.92 1.93 0.23 1.63 – 2.23 ON 3.55 3.58 0.28 3.12 – 3.89

OS 2.16 2.11 0.42 1.66 – 2.67 OS 3.68 3.73 0.40 3.14 – 4.12

OV 2.40 2.37 0.37 2.07 – 2.81 OV 4.10 4.17 0.55 3.25 – 4.67

LL OA 12.12 12.10 0.81 11.16 – 12.80 WPB OA 3.32 3.28 0.29 2.95 – 3.76

OP 12.01 11.75 1.21 10.43 – 13.68 OP 3.31 3.35 0.29 2.82 – 3.75

OB 13.76 13.83 0.87 12.61 – 14.98 OB 4.35 4.30 0.39 3.91 – 4.84

OE 17.67 17.54 0.94 16.67 – 18.93 OE 5.20 5.10 0.56 4.55 – 5.98

OG 12.00 12.07 1.23 10.20 – 13.44 OG 3.35 3.38 0.41 2.86 – 3.90

ON 10.90 11.11 0.85 9.91 – 11.94 ON 3.18 3.23 0.26 2.80 – 3.52

OS 11.95 11.63 1.34 10.72 – 14.10 OS 3.24 3.26 0.39 2.75 – 3.74

OV 15.33 15.50 1.01 13.92 – 16.51 OV 4.31 4.35 0.60 3.53 – 5.10

WL OA 9.11 9.15 0.54 8.31 – 9.76 WYM OA 0.24 0.19 0.24 0.00 – 0.67

OP 10.38 10.21 0.75 9.39 – 11.32 OP 1.12 1.10 0.32 0.72 – 1.32

OB 12.07 12.02 0.98 10.91 – 13.45 OB 1.41 1.67 0.99 0.00 – 2.63

OE 14.20 14.05 0.97 13.00 – 15.46 OE 0.19 0.00 0.23 0.00 – 0.53

OG 9.55 9.55 0.85 8.55 – 10.72 OG 0.12 0.00 0.17 0.00 – 0.35

ON 9.82 9.99 1.01 8.41 – 11.18 ON 2.58 2.66 0.45 2.02 – 3.07

OS 10.39 10.44 0.96 9.26 – 11.47 OS 2.58 2.57 0.47 1.96 – 3.28

OV 11.99 11.99 0.90 11.09 – 13.10 OV 0.09 0.00 0.17 0.00 – 0.38

LLL OA 9.07 9.13 0.65 8.26 – 9.83 MCL OA – – – –

OP 9.59 9.60 1.02 8.41 – 11.05 OP 0.19 0.22 0.180 0.00 – 0.43

OB 11.13 11.07 0.94 10.11 – 12.23 OB 0.56 0.63 0.38 0.00 – 1.02

OE 13.26 13.26 0.87 12.22 – 14.49 OE – – – –

OG 8.74 8.90 1.11 7.46 – 10.19 OG – – – –

ON 8.68 8.86 0.93 7.42 – 9.64 ON 0.70 0.64 0.235 0.46 – 1.04

OS 9.82 9.59 1.16 8.53 – 11.30 OS 1.08 0.84 0.89 0.37 – 2.70

OV 11.40 11.31 0.84 10.20 – 12.48 OV – – – –

WLL OA 2.48 2.61 0.37 1.95 – 2.97 DMS OA – – – –

OP 3.13 3.08 0.38 2.61 – 3.75 OP 0.00 0.00 0.00 0.00 – 0.00

OB 3.56 3.50 0.57 3.00 – 3.98 OB 0.43 0.44 0.42 0.00 – 1.00

(continued)



range would therefore be of great interest, especially

concerning the eastern Mediterranean taxa. This

would allow the occurrence of polyploidy in sect.

Pseudophrys to be assessed on a broader geographic

scale and could indicate the evolutionary importance

of this feature.

Ophrys eleonorae and O. vallesiana (O. iricolor group)

The cluster analyses (Figure 3), and the PCA

(Figure 4) clearly separated O. eleonorae and O.

vallesiana. These two taxa belong to the O. iricolor

group (Devillers & Devillers-Terschuren 2000c;

Table IV. (Continued).

Character Taxa Mean Median

Standard

deviation

10–90

percentile Character Taxa Mean Median

Standard

deviation

10–90

percentile

OE 3.86 3.90 0.39 3.39 – 4.37 OE – – – –

OG 2.58 2.54 0.38 2.13 – 3.04 OG – – – –

ON 3.02 3.00 0.34 2.60 – 3.42 ON 0.92 0.93 0.31 0.65 – 1.23

OS 3.17 3.16 0.43 2.70 – 3.74 OS 0.97 1.01 0.34 0.52 – 1.36

OV 3.31 3.27 0.38 2.95 – 3.75 OV – – – –

WCL OA 9.00 8.86 0.85 8.09 – 9.96

OP 6.33 6.34 0.54 5.60 – 7.08

OB 7.19 7.09 0.88 6.14 – 8.42

OE 9.00 8.86 0.85 8.09 – 9.96

OG 5.72 5.70 0.58 5.08 – 6.41

ON 5.16 5.21 0.66 4.31 – 5.78

OS 5.34 5.31 0.83 4.15 – 6.34

OV 7.48 7.62 0.72 6.39 – 8.27

Figure 3. Cluster analysis of Tunisian populations of sect.

Pseudophrys, based on 20 morphological characters (see Table

V A). For population codes see Table I.

Figure 4. Principal components analysis of Tunisian populations

of sect. Pseudophrys, based on 20 morphological characters (see

Table V. A). The first three axes accounted for 64.85%, 23.82%

and 5.11% of the total variation, respectively.

Table V. A. Principal component analysis of populations of

Tunisian Pseudophrys based on 20 morphological characters (see

Figure 4). Component loadings show contributions of the

characters to principal components (PC 1, PC 2). B. Canonical

discriminant analysis of populations of Tunisian Pseudophrys based

on 20 morphological characters (figure not shown). Total

canonical structure expressing correlation of morphological

characters with canonical axes (CAN 1, CAN 2, CAN 3) is

presented.

Character

A B

PC 1 PC 2 CAN 1 CAN 2 CAN 3

LS 0.866 0.478 70.045 0.047 0.803

WS 0.925 0.373 70.015 70.041 0.936

LP 0.852 0.502 70.148 70.029 0.832

WP 0.929 0.322 70.033 0.182 0.903

LL 0.859 0.505 70.137 70.206 0.853

WL 0.958 0.277 70.128 70.179 0.946

LLL 0.937 0.322 70.175 70.304 0.832

WLL 0.988 70.021 70.241 70.147 0.881

WCL 0.908 0.405 70.032 70.105 0.790

LSP 0.755 0.653 70.055 70.234 0.761

SPT 70.415 70.856 70.194 0.276 70.133

SPS 70.423 70.870 0.114 70.528 0.297

WLB 0.869 0.365 70.017 0.039 0.800

WPB 0.912 0.391 70.060 70.014 0.820

WYM 70.499 70.852 0.232 0.176 70.401

LL/WL 0.131 0.924 70.092 70.132 0.141

LL/LLL 0.067 0.946 0.347 0.488 0.059

LL/LSP 0.573 70.806 70.121 0.209 70.542

WL/LSP 70.507 70.852 70.082 0.221 70.546

WLB/WPB 70.895 70.348 0.591 0.628 70.498

54 F. Amich et al.


Figure 5. Variation in selected morphological traits of sect. Pseudophrys. Rectangles define 25 and 75 percentiles; circles show medians;

whiskers are from 10 to 90 percentiles. A, LL (mm); B, WL (mm); C, LLL (mm); D, ratio LL/LLL; E, LSP (mm); F, SPS (mm); G, WYM

(mm); H, ratio WLP/WPB.



Delforge 2002a, 2005), which is characterized by

crests at the base of the labellum that are obliquely

inclined outwards, to form a sort of tabletop. The

ratio between the WPB and the width of the labellum

base (WLP) is always 5 1 (Figure 5H); this clearly

distinguishes the O. iricolor group from the remaining

groups of the section Pseudophrys.

Paulus and Gack (1995, 1999) and Strohle (2003)

consider both taxa to be subspecies of O. iricolor

(O. iricolor subsp. eleonorae (Devillers-Tersch. &

Devillers) Paulus & Gack and O. iricolor subsp.

vallesiana (Devillers-Tersch. & Devillers) Paulus &

Gack, respectively), whereas Devillers and Devillers-

Terschuren (2000c) and Delforge (2002a) treat them

as species. Devillers and Devillers-Terschuren

(2000c) indicate that it is not coherent, on the basis

of morphological divergences, to treat some of the

taxa of the O. iricolor group at the species rank and

others at the subspecies rank.

Ophrys eleonorae was described from Sardinia by

Devillers and Devillers-Terschuren (Delforge et al.

1991), and reported to have a North African and

Cyrno-Sardian distribution. Ophrys vallesiana was

described from Tunisia (Tunis, Sidi Thabet, Djebel

Ahmar) by Devillers and Devillers-Terschuren (1994),

Figure 6. Cluster analysis of Tunisian populations of the Ophrys

subfusca group, based on 22 morphological characters (see Table

VI A). For population codes see Table I.

Figure 7. Principal components analysis of Tunisian populations

of the Ophrys subfusca group, based on 22 morphological

characters (see Table VI A). The first three axes accounted for

66.82%, 12.80% and 7.45% of the total variation, respectively.

Figure 8. Canonical discriminant analysis of individuals of

Tunisian representatives of the Ophrys subfusca group, based on

22 morphological characters (see Table VI B). The two axes

explain 79.14% and 9.14% of the total variation, respectively.

Table VI. A. Principal component analysis of populations of

Tunisian members of the Ophrys subfusca group, based on 22

morphological characters (see Figure 7). Component loadings

show contributions of the characters to principal components (PC

1, PC 2, PC 3). B. Canonical discriminant analysis of individuals

of Tunisian members of the Ophrys subfusca group, based on 22

morphological characters (see Figure 8). Total canonical structure

expressing correlation of morphological characters with canonical

axes (CAN 1, CAN 2, CAN 3) is presented.

Character

A B

PC 1 PC 2 PC 3 CAN 1 CAN 2 CAN 3

LS 0.947 70.012 70.041 70.154 0.188 0.459

WS 0.911 0.231 0.306 70.178 0.330 0.299

LP 0.846 70.211 70.211 70.036 0.202 0.654

WP 0.931 0.042 0.261 70.065 0.315 0.349

LL 0.952 0.243 0.141 70.301 0.474 0.334

WL 0.856 0.291 0.412 70.282 0.407 70.026

LLL 0.962 0.105 0.182 70.225 0.484 0.342

WLL 0.730 0.252 0.539 70.015 0.156 70.065

WCL 0.753 0.524 0.352 70.301 0.183 70.178

LSP 0.936 0.276 0.127 70.303 0.440 0.006

SPT 70.836 70.139 0.032 0.090 70.149 0.083

SPS 0.123 70.926 70.212 0.137 0.573 0.575

WLB 0.792 0.420 0.398 70.371 0.506 70.078

WPB 0.784 0.399 0.448 70.406 0.619 70.189

WYM 70.627 70.718 70.177 0.497 0.518 70.075

LL/WL 0.316 70.018 70.867 70.041 0.021 0.370

LL/LLL 70.397 0.522 70.261 70.034 70.138 70.091

LL/LSP 70.909 70.240 70.074 0.184 70.315 0.127

WL/LSP 70.925 70.202 0.095 0.163 70.305 70.035

WLB/WPB 70.373 70.119 70.624 0.099 70.200 0.224

MCL 70.429 70.675 70.005 0.154 0.296 70.101

DMS 70.547 70.762 70.148 0.298 0.347 70.082

56 F. Amich et al.


and is reported to be endemic to that country. Both

taxa coexist in Tunisia (Hervouet & Hervouet 1998).

Although the two taxa of the Ophrys iricolor group

are very similar, the recognition of O. eleonorae and

O. vallesiana as distinct taxa is clearly supported by

morphometric analysis. Both taxa are characterized

by a number of morphological traits (see Table IV;

Figure 5), the most important being the size of the

labellum, 4 (16.5–)17 mm in O. eleonorae and 5 16

(– 16.5) mm in O. vallesiana. At the same time, they

are clearly differentiated from the other Tunisian

taxa of sect. Pseudophrys.

In the present work the two studied members of

the O. iricolor group are accepted as distinct species.

Given their morphological and geographical differ-

ences from O. iricolor Desf., it is believed inappropri-

ate to treat them as subspecies.

Ophrys africana and O. gazella (O. fusca group)

Neither the cluster analyses (Figure 3) nor the PCA

(Figure 4) separated these two taxa, which belong to

the Ophrys fusca group (Delforge 2002a, 2005).

Ophrys gazella was described from Tunisia by

Devillers and Devillers-Terschuren (2000b); the

authors ascribed it a distribution across the regions

between Tunis and Bizerte. This taxon was formerly

referred to as ‘‘Ophrys fusca forme 1’’ by Valles and

Valles-Lombard (1988). Ophrys africana, a contro-

versial taxon from the northeastern part of Tunisia

(Cap Bon), was described by Foelsche and Foelsche

(2001), who compared it with similar taxa from

Tunisia, especially O. gazella. Based on a morpho-

logical and phenological study of both taxa, Delforge

(2002b) considered O. africana a later synonym of

O. gazella. However, Strohle (2003) continues to

treat them as distinct species.

Ophrys africana is distinguished from O. gazella by

its generally longer labellum (10–14 mm and 9–

12 mm, respectively) and earlier phenology. However,

the majority of quantitative traits studied in the present

work show it to fall clearly within the range of

variability of O. gazella (Table IV; Figure 5). No

consistent separation of the two taxa seems possible.

The above findings concerning O. gazella, when

seen in conjunction with the findings of Bernardos

et al. (2005) concerning O. bilunulata populations of

the Iberian Peninsula, suggest that O. gazella and O.

bilunulata may in fact be a single species. Before their

description of O. gazella (Devillers & Devillers-

Terschuren 2000b), Devillers and Devillers-Terschu-

ren (1994) considered the small-flowered Tunisian

plants they examined from Cap Bon and the Moun-

tains of Teboursouk (which clearly belonged to the O.

fusca group) to be O. bilunulata Risso. The only

distinguishing trait mentioned by Delforge (2005) for

these taxa, the angle formed by the outer edge of the

lateral lobe of the labellum and the longitudinal axis of

the labellum (35–458 in O. gazella, 22–358 in O.

bilunulata), was highly variable in the studied popula-

tions. Further detailed studies are required to decide

whether these taxa should be merged.

O. subfusca group

Neither the cluster analyses (Figure 6), the PCA

(Figure 7) nor the CDA (Figure 8) clearly separated

the putative species of this group.

This group is very complex and taxonomically

controversial, its members showing characteristics

intermediate between the O. fusca and O. lutea

groups. A consistently yellow labellum margin of

variable width distinguishes the O. subfusca

group from the O. iricolor and O. fusca groups

(Figure 5G). Delforge (2002a, 2005) includes five

North African species in this group, whereas

Devillers and Devillers-Terschuren (2000a) only

admit four, although they indicate that further

Figure 9. Variation in selected morphological traits of Tunisian representatives of the Ophrys subfusca group. Rectangles define 25 and 75

percentiles; circles show medians; whiskers are from 10 to 90 percentiles. A, MCL (mm); B, DMS (mm).



species might exist in North Africa, especially in the

mountains of Algeria.

In the present work, all the species accepted by

Devillers and Devillers-Terschuren (2002a) were

studied: O. aspea, O. battandieri, O. numida and O.

subfusca. Ophrys aspea is a Tunisian endemic, O.

battandieri (¼O. lutea subsp. murbeckii (H.

Fleischm.) Soo, sensu Strohle 2003)) and O. subfusca

(¼O. fusca Link, sensu Strohle, 2003) are North

African endemics (Algeria and Tunisia), and O.

numida is found in North Africa, Sicily and Malta.

The fifth North African species recognized by

Delforge (2002a), O. pectus Mutel (¼O. fusca Link,

sensu Strohle 2003), was not included in the present

analysis because of the difficulty in reliably identify-

ing this taxon; there is no way to clearly distinguish

Figure 10. Microphotographs of Ophrys iricolor and O. fusca groups chromosomes. Scale bars¼ 5 mm. A, O. eleonorae, sample OE02/05,

2n¼36, mitotic prophase; B, O. eleonorae, sample OE04/05, 2n¼ 36, mitotic prophase; C, O. vallesiana, sample OV22/05, 2n¼ 36, mitotic

prophase; D, O. vallesiana, sample OV65/05, 2n¼36, mitotic prophase; E, O. gazella, sample OG32/05, 2n¼36, mitotic prophase;

F, O. gazella, sample OG10/05, 2n¼ 36, mitotic prophase.

58 F. Amich et al.


it, and it has been interpreted differently by different

authors (i.e. Devillers & Devillers-Terschuren

2000a; De Belair et al. 2005).

The current tendency to attribute taxonomic

significance to obscure differences in morphological

floral traits, phenology or pollinating agents has led

to the extremely fine splitting of this group. How-

ever, the present analysis suggests that morphological

variation in the group is highly complex.

Ophrys aspea is well characterized by the brown

hairs on its labellum. The indumentum surrounds the

apex of the sinus, and extends to, or almost to, the

Figure 11. Microphotographs of Ophrys subfusca group chromosomes. Scale bars¼ 5 mm. A, O. aspea, sample OP25/05, 2n¼36, mitotic

prophase; B, O. battandieri, sample OB21/02, 2n¼36, mitotic prophase; C, O. battandieri, sample OB70/05, 2n¼36, mitotic prophase;

D, O. numida, sample ON44/05, 2n¼36, mitotic prophase; E, O. subfusca, sample OS15/05, 2n¼36, mitotic prophase; F, O. subfusca, sample

OS52/05, 2n¼36, mitotic prophase.



labellum’s edge. The yellowish margin is always

51.5 mm wide (Table IV; Figures 5 and 9).

Ophrys battandieri has a number of traits that allow it

to be distinguished from the rest of the group, mainly

involving the size of its labellum and speculum: LL,

WL, LLL, LSP (Table IV; Figures 5 and 9).

With respect to O. numida and O. subfusca, the

morphological transitions of the labellum in terms of

colour and pilosity make the distinction of these

species questionable, although the size of the

labellum (5–9 (710) mm in O. subfusca, and 8–

11.5 mm in O. numida) has sometimes been used

(Delforge 2005). However, the values for several

dimensions were found to overlap in the present

work (Table IV; Figures 5 and 9). In addition, the

values for the majority of floral traits examined in

O. numida fell clearly within the range of variability of

O. subfusca (Table IV; Figure 5). The existence of

characters reliably distinguishing O. subfusca from

O. numida seems unlikely.

The species of the O. subfusca group differ by few

but relatively well-marked characteristics. Therefore,

a generalization of the subspecies rank to all the taxa

of the group (e.g. Kreutz 2004) is of little use,

provides no information, and is undesirable given the

existence of sympatric forms. Therefore, we have

decided to accept the three taxa of the O. subfusca

group studied as distinct species.

The evidence presented in this paper suggests that,

given the generally weak morphological differences

between the studied taxa, morphological traits are

insufficient to support the taxonomic validity of all

these species, at least in some groups. Rather they

suggest that phenological, karyomorphological, eco-

geographical and molecular traits should be taken into

account if the plasticity and genetically determined

morphological variation is to be disentangled and

more fully resolved and understood. Thus, the

relationships among Pseudophrys taxa are to be more

fully appreciated and species diversification better

understood. Differences in flowering phenology could

form an effective barrier to gene exchange, which

might explain the existence of taxa that are apparently

strongly related from a morphological point of view.

Due to the rarity and narrow distribution area of

some of the species studied in this work, a thorough

knowledge of its reproductive system will be im-

portant for developing strategies for species manage-

ment and conservation, as shown in other taxa (e.g.

Jersakova & Kindlmann 2004; Mateu-Andres &

Segarra-Moragues 2004).

The results of the present study support the

recognition of six species that can be identified

according to the following key:

Key to the Tunisian representatives of the studied taxa of

sect. Pseudophrys

1. Labellum with a yellow margin (1.25–)1.5–2.5

(73) mm wide, with a crown of yellow hairs

reaching the edge (Ophrys subfusca group) .......4

1. Labellum without these characteristics, without

yellow margin or with a yellow margin of less

than 1.25 mm ................................................2

2. Base of the labellum with crests inclined

obliquely outwards, forming a sort of tabletop

(Ophrys iricolor group).....................................3

2. Base of the labellum with no flat area (Ophrys

fusca group)....O. gazella (including O. africana)

3. Labellum longer than (16–) 17 mm ..................

.................................................... O. eleonorae

3. Labellum shorter than 16 mm .......O. vallesiana

4. Brown hairs at the centre of the labellum

surrounding the apex of the sinuses ..... O. aspea

4. Labellum hairs separated from the apex of the

sinuses by a yellow margin .............................5

5. Labellum hairs reaching the edge of the

labellum; labellum 13–146 11.5–12.5 mm.......

.................... O. subfusca (including O. numida)

5. Labellum with a glabrous margin; labellum 11–

12.56 9–11 mm ..........................O. battandieri

Acknowledgements

The authors thank Dr H.A. Pedersen (Botanical

Garden & Museum, Natural History Museum of

Denmark, University of Copenhagen) for his very

valuable comments and constructive criticism of the

first version of the manuscript. We also thank Dr D.

Tyteca, P. Delforge, E. Vela and J. Viglione for their

valuable discussions, company and assistance in the

field in Tunisia during the botanical trip of 2002, and

for indicating suitable study populations. The authors

are especially thankful for the useful comments of the

anonymous reviewers. Financial support was pro-

vided by Spain’s Ministerio de Educacion y Ciencia

(project CGL2006-01600/BOS) and Comunidad

Autonoma de Castilla y Leon (project SA060A07).

The second author is supported by a research grant

co-financed by the European Social Fund and the

Junta de Castilla y Leon (Spain).

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