Revue internationale d'écologie méditerranéenne International ...

LAURENT HARDION, ALEX BAUMEL, PIERRE-JEAN DUMAS, NATHALIE DUONG, LAURENCE AFFRE & THIERRY TATONI
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Introduction
With about 3000 species divided into 245 taxonomic
sections, Astragalus L. (Fabaceae) can
be considered as the largest genus of flowering
plants (Podlech 1998). This genus mainly
present in Northern hemisphere, shows several
diversity poles as Western-North America
(400 spp.), the Andes (100 spp.), and the rich
South-Western and Sino-Himalayan regions
of Asia (1500-2000 spp.), supposed to be the
geographic origin of Astragalus (Wojciechowski
2005). This genus also occurs in
the Mediterranean Basin under different forms
and ecological conditions (Podlech 2008).
Within the Western part of the basin most of
taxa are only European species and among
them, Astragalus tragacantha L. (syn. A. massiliensis
Miller) shows unusual particularities
(Podlech 1999). This shrub is a thorny cushion
plant with spiny rachises which persist
after leaflets fall to reduce transpiration. It is
a member of subgenus Cercidothrix Bunge
(section Tragacantha DC). However, majority
of thorny cushion taxa belong to subgenus
Tragacantha Bunge, some of which are found
in Europe and majority are in South-Western
Asia and Sino-Himalayan region (Tutin et al.
1968). Moreover, this species is a protected
rare West-Mediterranean endemic with a fragmented
distribution, partly depending on its
ecology, from Portugal to Italy, Corsica and
Sardinia included (Valsecchi 1994). This
species takes refuge nearby the seashore, up
to 300 meters of altitude, on rocky soils or
sands, where strong winds favor prostrate
plants. Other West Mediterranean astragals of
section Tragacantha with similar morphological
characters seem to be close parents of A.
tragacantha (Nimis 1981). They are located
on Mediterranean islands (Baleares, Corsica,
and Sardinia) or in Southern Europe. But contrary
to A. tragacantha, they are orophyte
species (Bacchetta & Brulo 2006).
Several systematic studies have been carried
out on this genus (Bunge 1868; Barneby
1964; Podlech 1982), but the difficulty of
finding discriminant morphological characters
for anatomic classifications makes it perilous
and unstable (Podlech 1986). Indeed, more
recent studies based on phylogenetic analyses
have shown that many taxonomical units are
paraphyletic (Wojciechowski et al. 1999;
Kazempour Osaloo et al. 2003). Thus, a
molecular phylogenetic approach is necessary
to improve the systematic of A. tragacantha.
Molecular works have been undertaken for
more than a decade on genus Astragalus
(Wojciechowski et al. 1999; Kazempour Osaloo
et al. 2005) and an important collection of
nucleotidic sequences have been constituted
in GenBank data base. Our objective consists
of positioning A. tragacantha into a generic
phylogeny based on nuclear ribosomal DNA
(nrDNA) internal transcribed spacers (ITS).
Additionally, this study further compare A.
tragacantha to its relative species on a set of
biological characters available from literature.
Material and methods
Taxon sampling
For the present analysis, Astragalus tragacantha
was sequenced from six samples for
nrDNA ITS, four coming from coastline near
Marseille, and two from South Corsica, near
Bonificao (appendix 1). Other ITS sequences
were obtained from GenBank, and chosen to
represent at best phylogenies already built
(Wojciechowski et al. 1999; Kazempour Osaloo
et al. 2003), with respect for clades
defined by Wojciechowski et al. (1999). A
part of taxon sampling was taken from
sequences mostly similar to A. tragacantha
ITS according to a BLAST analysis. Finally
49 nr DNA ITS sequences of Astragalus were
used to build a phylogeny of this genus. Gen-
Bank accession numbers of these sequences
are referred in appendices 1 and 2. Considering
that most of astragal subgenera and sections
based on anatomic characters are paraphyletic
(Kazempour Osaloo et al. 2003), this
study limits their use or cites Maassoumi
modified taxonomy (Maassoumi 1998).
DNA extraction, PCR
and DNA sequencing
Leaves and stems of A. tragacantha were
dried with silica gel and ground in liquid
nitrogen before performing DNA extraction.
Total DNA was extracted and precipitated in
a 0.6 M Sodium acetate ethyl alcohol solution
and washed with 70% ethyl alcohol before
being resuspended in pure H 2 O. The internally
transcribed spacer 1, the 5.8S gene and
the internally transcribed spacer 2 were
amplified with primers ITS1 (TCCGTAGGT-
GAACCTGCGG) and ITS4 (TCCTCCGCT-
TATTGATATGC). PCRs were carried out in
ecologia mediterranea – Vol. 36 (1) – 2010