(a) Multidivtime analysis
We followed the protocols described by Rutschmann (2004). The nucleotide
frequencies, transition / transversion ratio and rate heterogeneity between the sites for each
dataset were calculated with Baseml, implemented in PAML (Yang 1997). These values
parameterize the F84 model, which is the most complex model implemented in Multidivtime
(Thorne & Kishino 2002). Using this model we calculated the branch lengths using
Estbranches, a component of Multidivtime, which were then rate corrected with Multidivtime.
The Markov Chain Monte-Carlo was run for 10,000 generations, retaining every 100th
sample, after discarding the first 100,000 generations. We repeated this twice, and compared
the results to ensure that stationarity had been reached. The dating was calibrated to the mean
dates obtained by the global analysis (described below). The standard deviations for rttm and
rtrate were set as equivalent to rttm and rtrate. Rttm was set to the estimate age of the basal
node, with each time unit equivalent to 10 Myr. Estbranches was used to obtain a tree with
estimated branch lengths. From this, we estimated the median amount of evolution between
the root and all the tips of the ingroup. Rtrate was set to this amount, divided by rttm (the
number of time units from the base to the tip of the tree). Bigtime was set to approximately
double the estimated age at the basal node.
(b) Global analysis and calibration
We built a phylogenetic tree for the Angiosperms, in which each study group was
represented by two species, selected to span the basal node of the study group. Further species
representing other groups as well as nodes for which fossils are available were also included.
The topology of the tree was taken from the three-gene Angiosperm phylogeny (Soltis et al.
2000). The tree was calibrated by the first occurrence of tricolpate pollen 125 Myr ago
(Anderson et al. 2005), the first occurrence of African Restionaceae in the 61 Myr old Banke
deposits in South Africa (Linder et al. 2003), the genistoid legume fossils reported by Lavin et
al. (2006) from 56 Myr ago, and the estimated age of the genus Phylica to 12 Myr based on
the age of St Helena (Richardson et al. 2001a; Richardson et al. 2001b). The tricolpate pollen
and the Phylica node were used as absolute dates (upper and lower bounds), while the other
two fossil deposits were used as lower bounds only. We used rbcL as “dating gene”, as
sequences of this gene were available for all the study groups, and as its semi-clocklike
behaviour has been well documented (Gaut et al. 1992). The sequences were largely
downloaded from Genbank, some were obtained from various researchers working on the
Cape flora. The sources are available as electronic supplementary material (see Table 1 of
electronic supplementary material).
The Disa tree was additionally calibrated using D. borbonica, an endemic of Réunion.
An upper age limit of 2 Myr was used, based on the age of Réunion (McDougall 1971). This
assumes that speciation occurred as the result of dispersal to Réunion. An underestimation of
the age of D. borbonica would occur with either of the following two scenarios: If the
mainland sister species of D. borbonica went extinct; alternatively if D. borbonica originated
elsewhere, migrated to Réunion and then went extinct in the source area. However, these
latter two scenarios are less parsimonious, and unlikely considering the short time-scales.
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