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

Additional file 2:

Theory of Planned Behaviour, Sun-safety questionnaire. (DOCX 28 kb

Additional file 3:

Theory of Planned Behaviour, Vitamin D questionnaire. (DOCX 26 kb

Additional file 1:

Study questionnaire (Socio-demographics, Knowledge and Stages of Change). (DOCX 89 kb