Supporting Information Methods S1 and Fig. S1
Methods S1 DNA extraction and sequencing and phylogenetic analyses
The systematic relations of Tasmannia are uncertain. Previously, many of the taxa
investigated were described as ‘entities’ of Drimys piperita (Vink, 1970). Entities have
no systematic nomenclatural meaning. Previously, Smith (1943) described several of the
entities studied here as Tasmannia species. Extensive molecular phylogenetic and
morphological evidence supports the distinction of Tasmannia from Drimys and provided
evidence that all of the Australian entities of D. piperita were Tasmannia species
(Raleigh et al., 1994; Doust & Drinnan, 2004; Marquínez et al., 2009; Worth et al.,
2010). Based on our results on the phylogenetic relations of New Guinean Tasmannia
and morphological studies (Vink, 1970), a case can be made for recognizing the original
species' designations of Smith (1943) and the recognition of new species from some of
the entities discussed earlier (such as ‘T. cordata’ and ‘T. nettioti’; Vink, 1970). Formal
descriptions of these taxa will be provided elsewhere.
Leaves of New Guinean Tasmannia were dried in silica gel. Sequences for other
species of Tasmannia as well as out-groups (Bubbia, Drimys, Pseudowintera,
Takhtajania, and Zygogynum) were taken from GenBank for phylogenetic analysis.
Tasmannia membranea and T. vickeriana were omitted from the phylogenetic analysis
because the DNA could not be isolated.
DNA was prepared with the Dneasy Plant Minikit (Qiagen, Valencia, CA, USA).
ITS amplifications were performed in 20 μl reactions using 10–20 ng of genomic DNA,
10X PCR buffer (Promega), 1.8–2.25 mM MgCl2, 0.2 mM each dNTP, 1.25 units of Taq
polymerase, and 0.2 μM each primer. Primers used were ‘ITS-4’ (5'TCCTCCGCTTATTGATATGC-3') and ‘ITS-5’ (5'GGAAGTAAAAGTCGTAACAAGG-3'). PCR was performed with the ‘ETS’ protocol.
All PCR products were checked by agarose gel electrophoresis. Sequences were prepared
utilizing the ABI Prism Dye Terminator Cycle Sequencing reaction kit and run at the
University of Tennessee Sequencing Facility (ABI 3100 DNA sequencer, Perkin Elmer
Inc., Foster City, CA, USA). Sequence alignment was performed manually. GenBank
accession numbers are provided in Table 1.
Phylogenetic analyses were performed with parsimony and Bayesian analyses
using PAUP and MrBayes 3.1.2, respectively (Ronquist & Huelsenbeck, 2001; Swofford,
2003). For parsimony, a heuristic search with 1,000 random addition replicates with TBR
branch-swapping was used, with gaps treated as missing data. Bootstrap analysis was
performed with 1000 replicates using a ‘Faststep’ search. Bayesian analysis was run for a
million generations with four chains and trees saved every 1000 generations. The number
of trees to discard as ‘burn-in’ was assessed by plotting likelihoods of and discarding
trees prior to the stable likelihood plateau. An appropriate maximum likelihood model of
sequence evolution (GTR+I+G; General Time Reversible model with a proportion of
invariant sites and gamma distributed rates) for the Bayesian analysis was chosen using
Modeltest (Posada & Crandall, 1998).
Divergence time estimates were obtained using a previously published approach
to focus on the nodes containing the Australian and Papua New Guinean samples
(Marquínez et al., 2009). Confidence intervals of ages (a = 0.05) were calculated
following Sanderson and colleagues (2004), by using 100 non-parametric bootstrap
replicates of the datasets using Seqboot (Phylip 3.68; Felsenstein, 1993) and running each
bootstrap using MrBayes. Two of the calibration points utilized by Marquínez et al.,
(2009) for the divergence of Takhtajania from the rest of the Winteraceae (77 mya and
92.4 mya) were used to infer divergence times of specific nodes in the Bayesian trees
obtained from ITS analyses.
Results
A low sequence divergence was found across the New Guinean Tasmannia taxa. The two
samples of T. cordata did not differ, and these samples differed by a minimum of four
substitutions from all other taxa. Other New Guinean Tasmannia differed from
previously reported T. piperita by 2–4 four base pairs (bp), and from T. cordata by 5–8
bp. Within Tasmannia, the basal-most divergences included Australian species, T.
lanceolata and T. insipida, respectively (Supporting Information Fig. S1). Above these, a
trichotomy consisting of two Australian clades (T. purpurascens +T. stipitata and T.
xerophila+T. glaucifolia) and a third clade of New Guinean taxa was reconstructed. The
two samples of T. cordata formed a basally diverging branch. The next split separated the
sample of T. piperita from the remainder of the New Guinea samples; four of the latter
were identical to one another (T. crassipes, T. heteromera, T. nettioti, and T. subalpina),
and the fifth, T. coriacea, differed by five substitutions. The estimated divergence time
for New Guinea clade samples of Tasmannia from the Australian ones was 29–34.9 mya
(Fig. S1).
Fig. S1 Phylogenetic relations and estimated molecular divergence estimates for
Australian and New Guinean Tasmannia taxa. The tree depicted is the consensus of
Bayesian analysis for ITS sequences. Estimated ranges for divergence ages are below
each node, and bootstrap levels of support denoted above each node.
References
Felsenstein J. 1993. Phylogeny Inference Package (PHYLIP). Version 3.5. Seattle, WA,
USA: University of Washington.
Ronquist F, Huelsenbeck JP. 2003. MRBAYES 3: Bayesian phylogenetic inference
under mixed models. Bioinformatics 19: 1572–1574.
Swofford DL. 2003. PAUP* 4.0b10. Sinauer Press.