Revue internationale d'écologie méditerranéenne International ...
Revue internationale d'écologie méditerranéenne International ...
Revue internationale d'écologie méditerranéenne International ...
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Phylogenetic relationships and infrageneric classification of Astragalus tragacantha L. (Fabaceae), inferred from nuclear ribosomal<br />
DNA Internal transcribed spacers data (nrDNA ITS)<br />
Appendix 1 – Subgenus, sections, geographic origins, voucher accessions and GenBank accession numbers<br />
of ITS sequences for species of Clade C.<br />
Species Subgenus Section Geographic origin; Voucher accession Genbank accession<br />
A. tragacantha Ga1 Cercidothrix Tragacantha Coastline, Frioul Island, Marseille, France; FRI2 GU223897<br />
A. tragacantha Ga2 Cercidothrix Tragacantha Coastline, Frioul Island, Marseille, France; FRI5 GU223898<br />
A. tragacantha Ga3 Cercidothrix Tragacantha Coastline, Cap Sicié, Var, France; AMP2 GU223899<br />
A. tragacantha Ga4<br />
A. tragacantha Co1<br />
Cercidothrix Tragacantha Coastline, Marseilleveyre, Marseille, France; MSV3 GU223900<br />
(A. trag. ssp. terraccianoi)<br />
A. tragacantha Co2<br />
Cercidothrix Tragacantha Coastline, Pertusiano, Bonifacio, Corse; PER5 GU223901<br />
(A. trag. ssp. terraccianoi) Cercidothrix Tragacantha Coastline, Pertusiano, Bonifacio Corse; PER12 GU223902<br />
A. fragrans Cercidothrix Acmothrix Iran; Maassoumi & Abouhamzeh 56916 (TARI) AB051967<br />
A. odoratus Cercidothrix Uliginosi Iran; Mozaffarian 64537 (TARI) AB051968<br />
A. uliginosus Cercidothrix Uliginosi China; NHQ124 EF685970<br />
A. falcatus Cercidothrix Uliginosi USA, CO, Boulder Co.; Weber 15359 U50488, U50489<br />
A. oreganus Cercidothrix Uliginosi USA, MT, Carbon Co.; McCarthy 107 AF121687<br />
A. canadensis var. brevidens Cercidothrix Uliginosi USA, NV, Nye Co.; W & S 133 U50496, U50497<br />
A. cymbicarpos Trimeniaeus Bucerates Spain; USDA 318945, W & S 410 AF121678<br />
A. hamosus Trimeniaeus Bucerates Iran; Maassoumi 47586 (TARI) AB051936<br />
A. edulis Trimeniaeus Bucerates Israel; USDA 244273, W & S 293 AF121677<br />
A. boeticus Trimeniaeus Cyamodes France; USDA 414243, W & S 300 AF121679<br />
A. glycyphyllos Phaca Glycyphyllus Iran; Assadi & Sardabi 24090 (TARI) AB051941<br />
A. depressus Phaca Tapinodes Germany; Podlech 39745 (TARI) AB231147<br />
100 µl volume containing 10 mM TrisHCL<br />
(pH 9), 50mM KCl, 1.75mM MgCl 2 ,<br />
0.6 mg/ml BSA, 0.1% Triton X100, 0.2mM<br />
of each primer, 0.4 mM dNTPs, 5 U Taq (Taq<br />
CORE Kit, Q-BIOgene) and 4 µl of DNA<br />
template at a concentration ranging from 126<br />
to 740 ng/ml. The PCR program was: 2 minutes<br />
at 94 o C, followed by 30 cycles (45 seconds<br />
at 94 o C, 45 seconds at 48 o C and 2 minutes<br />
at 72 o C), ending with 5 minutes at 72 o C.<br />
PCR products were directly sequenced by<br />
AGOWA services. Sequences were carefully<br />
checked on chromatograms with the MEGA<br />
4 software program (Tamura et al. 2007).<br />
Phylogenetic analyses<br />
Sequences were aligned by using ClustalW<br />
software (Thompson et al. 1994) implemented<br />
in Mega 4.0. Alignments were refined<br />
manually and each sequence required introduction<br />
of numerous single and multibase<br />
gaps, especially with out-group. Neighborjoining<br />
(NJ) tree was built in Mega 4.0 based<br />
on Kimura 2-parameter distances (Kimura<br />
1980). Gaps/missing data were treated with<br />
pairwise deletion option. To quantify the support<br />
of clades, bootstrap values were calculated<br />
from 5000 replicate analyses. Parsimony<br />
analyses were performed with PAUP*4<br />
(Swofford 2002). Heuristic search were used<br />
to analyze the 49 taxa. Based on previous<br />
ecologia mediterranea – Vol. 36 (1) – 2010<br />
Appendix 2 – GenBank accession numbers of ITS sequences for NJ tree,<br />
out of Clade C.<br />
A. williamsii AF121685<br />
A. australis AF121686<br />
A. siliquosus AB051940<br />
A. sinicus AB051965<br />
A. complanatus EU591995<br />
A. alpinus L10760<br />
A. lamprocarpus AB052015<br />
A. brachycalyx AB052026<br />
A. compactus AB231136<br />
A. cicer AB231110<br />
A. pulchellus L10786<br />
A. glaux AB231111<br />
A. austriacus AB231094<br />
A. echinatus AB051938<br />
A. oophorus AB051993<br />
studies, Melilotus alba was used arbitrarily as<br />
out-group (Wojciechowski 2005). Parameters<br />
of the search were the following: start = stepwise,<br />
addseq = random, nreps = 1000, randomize<br />
= addseq, swap = tbr, multrees = yes.<br />
An exhaustive search using branch and bound<br />
method was conducted on a subset of taxa<br />
related to Astragalus tragacantha, following<br />
the default parameters of PAUP 4. For both<br />
searches, constant and uninformative characters<br />
on one hand, and gaps/missing data on<br />
the other hand were removed from the data<br />
matrix. Support for the different clades was<br />
tested by bootstrap analysis (5000 replicates<br />
using heuristic search).<br />
A. patagonicus AF121746<br />
A. arizonicus AF121690<br />
A. chinensis FJ980292<br />
A. vulcanicus AB051960<br />
A. alopecias AB051949<br />
A. jessenii AB051950<br />
A. kirrindicus AB051951<br />
A. tawilicus AB051948<br />
A. dictyolobus AB051947<br />
A. yatungensis AF521955<br />
A. epiglottis AB051910<br />
Lessertia herbacea AF121753<br />
Oxytropis aucherii AB051908<br />
Oxytropis splendens AF121761<br />
Melilotus alba DQ006009<br />
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