Journal of Biogeography
SUPPORTING INFORMATION
Phylogeography and evolution of three closely related species of Tsuga (hemlock) from subtropical eastern Asia: further insights
into speciation of conifers
Yu-Zhi Cun and Xiao-Quan Wang
Appendix S2 Phylogenetic reconstruction and divergence time estimation for Tsuga species.
Figure S1 Fossil-calibrated molecular chronogram of the genus Tsuga based on combined trn S/fM+ atp H/I+ psb J/ pet A sequences. The red circle indicates the age-constrained node, numbers above the branches indicate substitution rates, numbers below the branches indicate the Bayesian posterior probabilities, and numbers on the right of the nodes indicate age estimates [mean ages (95% HPD intervals)].

Figure S2 Fossil-calibrated molecular chronogram of the genus Tsuga based on combined trn S/fM+ atp H/I+ psb J/ pet A sequences. Red and yellow circles indicate age-constrained nodes, numbers above the branches indicate substitution rates, numbers below the branches indicate the Bayesian posterior probabilities, and numbers on the right of the nodes indicate age estimates [mean ages (95%HPD interval)].
The divergence times of Tsug a species were estimated based on the combined cpDNA trn S/fM+ atp H/I+ psb J/ pet A sequences, with each species represented by one individual. Nothotsuga longibracteata was used as outgroup according to published phylogenies (Vining, 1999; Wang et al ., 2000; Havill et al ., 2008).
The rate constancy across lineages was evaluated using a likelihood ratio test
(LRT), in which likelihood scores of the trees with and without an enforced molecular clock were compared (Felsenstein, 1988). Since the clock assumption was rejected (
δ
= 145.81738, d.f. = 8, P < 0.001), divergence times and substitution rates were estimated with BEAST 1.7.5 (Drummond & Rambaut, 2007), using the HKY+G substitution model, an uncorrelated lognormal relaxed clock model, and the Yule model of speciation.
Two methods were tried to calibrate the molecular clock: (1) only the most recent common ancestor of Nothotsuga and Tsuga was fixed at an age of 90 Ma following Havill et al . (2008), based on the oldest pollen record of Tsuga from the
Late Cretaceous (c. 90 Ma) deposits in Poland (Macko, 1963); and (2) another calibration was added, and the crown age of Tsuga was set with a normal prior distribution of 47 ± 4.9 Ma, based on the earliest record of Tsuga megafossils from the
Eocene deposits (LePage, 2003), which is consistent with the crown age of Tsuga
(42.26 ± 8.02 Ma) estimated by Havill et al . (2008) based on another three cpDNA markers ( trn T/ trn F, trn Kmat K, rpl 16). We ran 20,000,000 generations of Markov chain Monte Carlo (MCMC), and sampled every 2000 generations, with a burn-in of
1000 trees. T RACER 1.5 was used to check convergence of chains to the stationary

distribution. The MCMC output analysis was conducted with T REE A NNOTATOR 1.7.5, and the chronological phylogeny was displayed by F
IG
T
REE
1.3.1.
The substitution rate (per site per year) of the T. chinensis
–
T. forrestii lineage was estimated to 0.28
× 10
-9
(95% HPD interval: 0.07-0.42
× 10
-9
) by using one calibration (Fig. S1) and 0.20
× 10 -9 (95% HPD interval: 0.05-0.41
× 10 -9 ) by using two calibrations (Fig. S2).
The phylogenetic relationships among Tsuga species were also inferred using
M R B AYES 3.1.2 (Ronquist & Huelsenbeck, 2003). Indels in the alignment were coded as 1/0 binary characters, and gaps of different lengths were all treated as single events.
The evolutionary model for the Bayesian analysis was determined by the hierarchical likelihood ratio test (LRT) using M ODELTEST 3.07 (Posada & Crandall, 1998), and the
F81 model (Felsenstein, 1981) with gamma-distributed rate variation (F81+G) was chosen. For the Bayesian inference, one cold and three incrementally heated Markov chain Monte Carlo chains were run for 1,000,000 generations and repeated twice to avoid spurious results. Trees were sampled every 100 generations with the first 300 samples discarded as burn-in. A 50% majority rule consensus tree was obtained based on those trees sampled after generation 30,000. The results showed that the 50% majority rule consensus tree was topologically consistent with that of the maximum clade credibility tree generated by
BEAST
1.7.5. Thus, the 50% majority rule consensus tree was not shown, but the Bayesian posterior probabilities were shown in the maximum clade credibility tree (Fig. S1).
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