Alignment of the sequences upload to Dryad_GYY_20141211

Alignment of the sequences upload to Dryad_GYY_2014121

Chronogram of slipper orchids inferred from the combined six chloroplast genes, and ancestral area reconstruction.

<p>The crown age of slipper orchids was set as a calibration point for time estimation. Two areas were defined: (A) Old World and (B) New World. The ancestral areas with the highest probabilitiy are shown above (S-DIVA) and below (Lagrange) the branches with pie charts.</p

SplitsTree_input file

SplitsTree_input fil

The distribution of slipper orchids modified from Pridgeon et al. [<b>165</b>].

<p>Shaded areas show the current species distribution, with different colors to represent the five genera. The tree topology indicates the phylogenetic relationships of slipper orchids reconstructed in this study.</p

Results of Model test and MrModel test.

<p>Results of Model test and MrModel test.</p

Estimated divergence times (Mya) derived from BEAST and r8s.

<p>Estimated divergence times (Mya) derived from BEAST and r8s.</p

Phylogenetic affinities and <i>in vitro</i> seed germination of the threatened New Zealand orchid <i>Spiranthes novae-zelandiae</i>

<p>The New Zealand “ladies’ tresses” <i>Spiranthes novae-zelandiae</i> is a terrestrial orchid currently classified as Threatened – Nationally Vulnerable. Although considered endemic to New Zealand, morphological similarity with the widespread Eurasian <i>Spiranthes sinensis</i> has cast doubt on its taxonomic and biogeographic status. Habitat destruction is the main threat to the survival of <i>S. novae-zelandiae</i>. Unfortunately, lack of information regarding its symbiotic fungal associates and technical expertise within New Zealand with symbiotic seed germination techniques have hindered its propagation from seed. In this study we examined the phylogenetic affinities of <i>S</i>.<i> novae-zelandiae</i> using nuclear (internal transcribed spacer) and chloroplast (<i>trn</i>L, <i>trn</i>S-G and <i>mat</i>K) DNA sequences. We also explored the diversity of fungal symbionts associated with <i>S. novae-zelandiae</i> and identified the fungal symbiont that promotes seed germination and seedling development using DNA sequences and <i>in vitro</i> seed germination experiments. Bayesian Inference analyses showed that <i>S. novae-zelandiae</i> is nested within <i>S. sinensis</i> along with <i>Spiranthes australis</i> and <i>Spiranthes</i> aff. <i>novae-zelandiae</i> (CHR 518297; Motutangi), a morphological variant of uncertain taxonomic status from northern New Zealand. These results support earlier suggestions that a broader concept of <i>S. sinensis</i> is needed to include <i>S. novae-zelandiae</i> and many other taxa segregated from <i>S. sinensis</i>. Nine fungal Operational Taxonomic Units were isolated from the roots of <i>S. novae-zelandiae</i> but only one promoted seed germination and seedling development. DNA sequence analyses confirmed that this isolate was a strain of <i>Tulasnella</i> (anamorph: <i>Epulorhiza</i>); a widespread mycorrhizal fungus previously found in <i>S. australis</i> and <i>S. sinensis</i>. Lastly, we describe the germination process and the steps we followed to obtain flowering plants after 2 years of seed inoculation.</p

The ML tree of slipper orchids constructed based on the combined cpDNA+nuclear genes.

<p>Numbers above branches indicate the bootstrap values ≥50% for the MP and ML analyses, respectively. Bayesian posterior probabilities (≥0.90) are shown in bold lines. Symbols on the right indicate the distribution of some important characters of slipper orchids.</p

Fossil-calibrated molecular chronogram of the family Orchidaceae based on combined <i>mat</i>K+<i>rbc</i>L sequences.

<p>Red circles indicate age-constrained nodes, and arrows indicate the crown ages of the five subfamilies of Orchidaceae.</p

Generation of <i>Tsc1^cko</i> mutant mice and characterization of <i>Tsc1</i> disruption by western blot and PCR analysis.

<p>(A) Schematic representation of deletion of <i>Tsc1</i> exon17 and exon18 by cyp19-cre mediated recombination in granulosa cells. (B) Granulosa cells were collected from COC of both <i>Tsc1^flox/flox</i> mice and <i>Tsc1^cko</i> mutant mice and lysed for western blot: <i>Tsc1</i> was almost absent in <i>Tsc1^cko</i> granulosa cells, β-Tubulin was used as an internal control. (C) PCR analysis indicated that cyp19-cre mediated recombination of <i>Tsc1</i> exclusively occurred in <i>Tsc1^cko</i> ovary.</p