Revision of Passiflora Subgenus Decaloba ... - Passion Flowers

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The ITS region for members of supersection Cieca was sequenced directly from
the cleaned amplified product in the DNA Sequencing Core Facility on the
University of Florida campus (DSEQ, UF) with ABI 377 and ABI 373A automated
sequencers. Sequences were edited and assembled using the ABI software
packages Sequence Navigator Version 1.0.1 and Auto Assembler Version
1.3.0 on an Apple PowerMac computer and aligned visually. Gaps were coded
as missing values.
Selected cleaned PCR products were cloned into TOPO-TA Cloning®
(Invitrogen, Carlsbad, California, USA) vectors according to the manufacturer’s
instructions, except that the ligation reactions were halved. Transformation
reactions were incubated in SOC broth (2.0% tryptone, 0.5% yeast extract,
10mM NaCl, 2.5mM KCl, 10mM MgCl2-6H2O, 20mM glucose) at 37°C for one
hour before being spread onto plates containing S-Gal/LB Agar/Kanamycin
Blend (Sigma, St. Louis, Missouri, USA) and incubated at 37°C for 8–18 hours.
Only large white colonies, representing potentially recombinant plasmids, were
selected for amplification and sequencing.
Phylogenetic Search Strategies
Two matrices were analyzed cladistically for this study: morphology (32 taxa
including outgroups) and ITS sequence data (71 taxa including outgroups). The
morphological character states were carefully delimited (see discussion under
“Morphological Data Set”). Many characters are qualitative, and discrete states
were delimited within quantitative characters by assessment of gaps in the
pattern of variation (Stevens, 1991). Multistate characters were considered to be
unordered and ingroup/outgroup relationships were analyzed simultaneously.