Page 3 - Passalacqua_Peruzzi_Pellegrino2008

TAXON 57 (3) • August 2008: 893–906 Passalacqua & al. • Biosystematics of the Jacobaea maritima group

analyses. One plant per population was cultivated under 2600) for 30 cycles. Conditions were as follows: 30 s de-
uniform conditions (open field) in the Botanic Garden, naturation at 94°C, 1 min annealing at 55°C, 45 s exten-
University of Calabria. sion at 72°C; extension time was increased by 3 s/cycle,
LEU
Molecular markers. — The trn LEU intron (cpDNA) with a final step of 7 min. PCR products were purified
and the Internal Transcribed Spacers (ITS) of ribosomal using Microcon microconcentrators MWCO 100,000
nuclear DNA were sequenced for three individuals of each (Amicon, Beverly, MA, U.S.A.) and sequenced in both
population. These regions were selected because they directions using a modification of the Sanger dideoxy
have been shown to be informative at the species level in method as implemented in a double stranded DNA cy-
Senecioneae (Alvarez-Fernandez & al., 2001; Comes & cle sequencing system with fluorescent dyes. Sequence
Abbott, 2001; Pelser & al., 2002; 2003). In addition, we reactions were then run on a 373A Applied Biosystems
also sequenced one herbarium specimen of J. ambigua Automated DNA sequencer (Applied Biosystems, Fos-
subsp. taygetea. ter City, CA, U.S.A.). The electropherograms of the in-
To evaluate genetic diversity among and within nat- vestigated specimens were aligned by using Sequences
ural populations of J. maritima s.l. and J. ambigua s.l. Navigator software (Perkin Elmer, U.S.A.). The maxi-
we used intersimple sequence repeats (ISSRs) markers. mum parsimony (MP) optimality criterion, as imple-
Previous studies have indicated that the ISSR technique mented in PAUP 4.0b10 (Swofford, 2002), was used
produces more reliable and more easily reproducible bands to infer phylogenetic relationships. Sequence limits of
LEU
than RAPDs, because of the higher annealing temperature the trn LEU intron, ITS I and ITS II were determined by
and longer sequence of ISSR primers (Tsumura & al., comparison with known sequences (Pelser & al., 2002,
1996; Nagaoka & Ogihara, 1997). 2003). Transitions and transversions were weighted
DNA extraction. — Leaf samples of 20 specimens equally. Bootstrap values were estimated from 1,000
were collected from each investigated population (Ta- replicates. In this case we used J. gigantea (sub S. gi-
ble 2) and preserved in silica gel until DNA extraction. ganteus: GenBank accessions AY155606, AY155617)
The J. taygetea accession included tissue samples from and J. vulgaris (sub S. jacobaea: GenBank accessions
herbarium specimens. DNA extraction was carried out AY155610, AY155621), both within the “S. jacobaea-
using a modified CTAB method (Doyle, 1991). The DNA clade” (Pelser & al., 2003), as outgroups. A distance
pellet was dissolved in 500 μl TE buffer solution. DNA matrix, prepared to resolve taxon divergence, was ob-
concentrations were estimated by agarose gel analysis tained with the Kimura two-parameter (K2P) method
stained with ethidium bromide using marker II (fagus-λ using PAUP 4.0b10 (Swofford, 2002). With regards to
digested with Hind III). the J. maritima group, we also included sequences of J.
LEU
Sequencing. — The chloroplast trn LEU intron and maritima from Spain and France (GenBank accessions
the Internal Transcribed Ribosomal Spacers (ITS I and AF459950, AF460158), of J. ambigua (taygetea) from
ITS II) were amplified by PCR, using specific primers Greece (GenBank accessions AF459927, AF460122)
(White & al., 1990; Taberlet & al., 1991). All PCR reac- and of J. gnaphalodes from Crete (GenBank accessions
tions were conducted in a thermal cycler (Perkin Elmer AY155607, AY155618) in our analysis.

Table 2. Taxonomic units and locality of investigated populations.
No. Taxonomic unit Locality
1 J. maritima Italy, Tuscany: Castiglioncello (Livorno), between loc. Forbici and loc. Pinze, 10 m a.s.l., 2.VI.2002,
Peruzzi
2 J. bicolor Italy, Sicily: Egadi Islands, Levanzo, along the road from the Port eastwards, 17.VI.2002, Peruzzi
& Passalacqua
3 J. bicolor Italy, Calabria: cliffs near Pizzo Calabro (Vibo Valentia), 19.VI.2002, Peruzzi & Passalacqua
4 J. bicolor Italy, Sicily: Eolian Islands, Lipari, at the caves of pumice, 18.VI.2002, Peruzzi & Passalacqua
5 J. bicolor Italy, Sicily: Cefalù, Capo Raisigerbi, in front of Valtur Village, 17.VI.2002, Peruzzi & Passalacqua
6 J. gibbosa Italy, Calabria: Bagnara Calabra (Reggio Calabria), near the Ruggero tower, 19.VI.2002, Peruzzi
& Passalacqua
7 J. gibbosa Italy, Sicily: Acqualandrone (Messina), near the fiumara, 18.VI.2002, Peruzzi & Passalacqua
8 J. ambigua Italy, Sicily: Nicolosi (Catania), foothills of Mount Etna, loc. I Tre Altarelli, ca. 700 m a.s.l., 16.VI.2002,
Peruzzi & Passalacqua
9 J. nebrodensis Italy, Sicily: Madonie, Quacella, 9 km along the road from Polizzi Generosa to Piano Battaglia, 16.VI. 2002,
Peruzzi & Passalacqua.

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