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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
To link to this Article: DOI: 10.1080/11263500802633485
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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
Sidi Thabet, Djebbes II, Jebel Ahmar, 224 m, 32SNF9781 (368520390 0N–108050260 0E),
17.3.2005, FA & SB
18 36 OG10/05 110682
OG11 TU SALA
Teboursouk, road to Thibar, 560 m, 32SNF1737 (368280410 0N–98110380 0E), 26.3.2005, FA & SB 10 – – 110633
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
Sidi Thabet, Djebbes II, Jebel Ahmar, 224 m, 32SNF9781 (368520390 0N–108050260 0E),
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
Teboursouk, road to Thibar, 560 m, 32SNF1737 (368280410 0N–98110380 0E), 26.3.2005, FA & SB 12 – – 110639
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
Teboursouk, road to Thibar, 560 m, 32SNF1737 (368280410 0N–98110380 0E), 26.3.2005, FA & SB 16 – – 110721
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.
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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
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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.
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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
Ophrys sect. Pseudophrys 51
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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.
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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)
Ophrys sect. Pseudophrys 53
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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.
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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.
Ophrys sect. Pseudophrys 55
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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.
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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).
Ophrys sect. Pseudophrys 57
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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.
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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.
Ophrys sect. Pseudophrys 59
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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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