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

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