Studies of Cryptostylis ovata and Microtis media orchids from Western Australia

Cryptostylis ovata is the only evergreen indigenous orchid that grows in south-western Australia; all the others are deciduous. It uses a classical ‘k’ strategy to reproduce, thus it has a lifespan of decades or longer, and although it produces large numbers of seed when its flowers are pollinated, it appears that only a tiny proportion germinate. On the other hand, Microtis media is the classical ‘r’ breeder in that it lives typically from only one to a few years, and its flower spike generates thousands of seed, which freely germinate. We studied mycorrhizal associations in C. ovata and M. media plants from different wild plants at five sites per species, and at different times throughout the year and life cycles of the plants. Fungi were isolated from pelotons in the roots of both species, and pure cultures were obtained. Diagnostic sequences were generated from PCR products using primers annealing to conserved sequences flanking variable internal transcribed spacer (ITS) regions of the nuclear ribosomal gene. These studies indicated that C. ovata plants from five different sites maintained relationships with Tulasnella fungi of four distinct taxa. The associations were collection site-specific and clustered into two groups - the southern and northern groups. Fungal isolates collected from three southern C. ovata populations closely resembled T. prima isolates identified from Chiloglottis sp. orchids. Fungal isolates identified from two northern orchid populations were of three undescribed Tulasnella taxa. M. media plants collected from disturbed and undisturbed sites also associated with Tulasnella-like fungi. These Tulasnella-like fungi are different from those associated in C. ovata plants. There were six Tulasnella species identified from M. media roots, in which four taxa were similar to previously-described Tulasnella species. Two other taxa were predicted to belong to the families Serendipitaceae and Ceratobasidiaceae. We attempted to germinate C. ovata seeds using different natural and artificial media, and with different mycorrhizal fungal partners. Seeds that germinated to stage 3 (appearance of protomeristem) only occurred on oatmeal agar (OMA) media and soil solution equivalent (SSE) media inoculated with a Tulasnella prima isolate identified from C. ovata orchids, but not with those identified from M. media plants. OMA was the most promising media for C. ovata seed germination; seeds plated on this media inoculated with fungi derived from M. media plants got to stage 1 of germination (imbibed embryo). Unfortunately, none of the treatments in this study reached the final stage of germination—the appearance of the second leaf. Morphological characteristics of above-ground organs of C. ovata plants were recorded. Plants in southern populations had many relatively large dark-green leaves and flowers while plants in the two northern populations had smaller and fewer pale-green leaves and no flowers during the study times. Genetic diversity of all or most plants of the five populations was assessed using inter simple sequence repeat (ISSR) markers. Genetic analysis revealed that plants within each population clustered together, and each population was genetically-distinct from the others. Our findings suggested that at two sites, all plants were genetically-identical, suggesting only asexual reproduction occurred there. At the other three sites, there were varying degrees of genetic diversity, suggestive of sexual reproduction. The most sexually-diverse population was one growing within a pine plantation where few other plants could colonise. We discuss the implications of our research in terms of reproductive strategies in C. ovata, and factors that may limit sexual reproduction