361Material and Methods
Phylogenetic Data
To estimate phylogenetic diversity we used the plastid and nuclear phylogeny of
Sarcolaenaceae produced by Haevermans et al. ( in prep ). The taxon sampling
includes 47 species belonging to the family, for a total of 91 Operational Taxonomic
Units (OTUs). All ten genera were represented and 66 % of the species were sam-
pled, thereby capturing most of the taxonomic and morphological/ecological diver-
sity and covering the full geographic distribution of Sarcolaenaceae.
In addition to the 47 Sarcolaenaceae sampled, 6 species were selected from their
sister group, Dipterocarpaceae, and 1 species from the next most-closely related
family, Cistaceae (Dayanandan et al. 1999 ; Ducousso et al. 2004 ; Haevermans et al.
in prep ), all of which served as outgroup taxa. Sequence data were obtained from
one nuclear (ITS) and three plastid ( rbcL, psbA - trnH , and psaA - ORF170 ) markers
(Haevermans et al. in prep ). We performed a Bayesian dating analysis using BEAST
v1.7.2 (Drummond et al. 2012 ) under the uncorrelated lognormal relaxed clock
model with a Yule prior on speciation. Data were partitioned according to the num-
ber of DNA regions and we applied to each partition the GTR+ I+G substitution
model, for reasons outlined by Huelsenbeck and Rannala ( 2004 ). An individual
MCMC run was conducted for 20 × 10^6 generations, with sampling every 1,000
iterations, thus generating 20,000 chronograms. We discarded the fi rst 25 % of sam-
ples as burn-in. Mixing of the chains and their convergence were verifi ed in Tracer
1.4 (Rambaut and Drummond 2007 ). Using Logcombiner, we merged the remain-
ing 15,000 trees and produced a maximum clade credibility (MCC) chronogram
using TreeAnnotator. We applied two temporal constraints to calibrate the tree, one
at the split between Dipterocarpaceae and Sarcolaenaceae based on Wikström et al.
( 2001 ), and another for the age of the stem-group of the clade comprising Leptolaena ,
Mediusella , Sarcolaena , Xerochlamys and Xyloolaena based on the estimated age
of a fossil pollen attributable to this group (Coetzee and Muller 1984 ).
In order to assess the cladogenesis process in Sarcolaenaceae , we measured the
degree of imbalance of the Sarcolaenaceae consensus tree topology using the R
package apTreeshape (Bortolussi et al. 2006 ), in conjunction with the R package
ape (Paradis et al. 2004 ). The imbalance was estimated by calculating the Colless’s
index (Mooers and Heard 1997 ). We compared this experimental value against
those obtained for 500 simulated trees built under the Equal Rate Markov (ERM)
Yule model or the PDA (Proportional to Distinguishable Arrangement) model in
which each tree is equally probable (Mooers and Heard 1997 ), using the function
colless.test () implemented in R package apTreeshape (Bortolussi et al. 2006 ). We
used the “less” and “greater” alternatives to test whether the tree is less unbalanced
or more unbalanced than predicted by the null model.
Conservation of Phylogenetic Diversity in Madagascar’s Largest Endemic Plant...