Shallow Genome Sequencing for Phylogenomics of Mycorrhizal Fungi from Endangered Orchids

ABSTRACT Most plant species form symbioses with mycorrhizal fungi and this relationship is especially important for orchids. Fungi in the genera Tulasnella, Ceratobasidium, and Serendipita are critically important for orchid germination, growth and development. The goals of this study are to understand the phylogenetic relationships of mycorrhizal fungi and to improve the taxonomic resources for these groups. We identified 32 fungal isolates with the internal transcribed spacer region and used shallow genome sequencing to functionally annotate these isolates. We constructed phylogenetic trees from 408 orthologous nuclear genes for 50 taxa representing 14 genera, 11 families, and five orders in Agaricomycotina. While confirming relationships among the orders Cantharellales, Sebacinales, and Auriculariales, our results suggest novel relationships between families in the Cantharellales. Consistent with previous studies, we found the genera Ceratobasidium and Thanatephorus of Cerabotasidiaceae to not be monophyletic. Within the monophyletic genus Tulasnella , we found strong phylogenetic signals that suggest a potentially new species and a revision of current species boundaries (e.g. Tulasnella calospora ); however it is premature to make taxonomic revisions without further sampling and morphological descriptions. There is low resolution of Serendipita isolates collected. More sampling is needed from areas around the world before making evolutionary-informed changes in taxonomy. Our study adds value to an important living collection of fungi isolated from endangered orchid species, but also informs future investigations of the evolution of orchid mycorrhizal fungi

Diversity and specificity of orchid mycorrhizal fungi in a leafless epiphytic orchid, Dendrophylax lindenii and the potential role of fungi in shaping its fine-scale distribution

Orchids grow in diverse habitats worldwide with most (approximately 69%) growing on trees as epiphytes. Although orchid mycorrhizal fungi have been identified as potential drivers for terrestrial orchid distribution, the influence of these fungi on the fine-scale distribution of epiphytic orchids is poorly understood. In this study, we investigated the mycorrhizal fungal community and fine-scale distribution of Dendrophylax lindenii, a rare and endangered epiphytic orchid that is leafless when mature. We used amplicon sequencing to investigate the composition of orchid mycorrhizal fungi in the roots of 39 D. lindenii individuals in their natural habitat, the swamps of Florida. We compared the orchid mycorrhizal fungi of D. lindenii to those of co-occurring epiphytic orchids, as well as to the orchid mycorrhizal fungal communities of bark from potential host trees, with and without D. lindenii. Our results show that D. lindenii has a high specificity for a single Ceratobasidium species, which is widely distributed on phorophytes and detected in both wet and dry periods in the orchid’s habitat. This Ceratobasidium species was mostly absent or only recorded in low frequency in the roots of co-occurring epiphytic orchids. Phylogenetic analysis documented that this Ceratobasidium was conspecific with the strain that is used to germinate D. lindenii ex-situ. However, our findings suggest that laboratory germinated adult D. lindenii transplanted into the field had lower read abundances of this Ceratobasidium compared to naturally occurring plants. These findings suggest that this orchid mycorrhizal fungus may play a significant role in the fine-scale distribution of naturally occurring D. lindenii

The orchid recovery program at Illinois College – a successful blend of teaching, research and undergraduate student participation to benefit orchid conservation

A decade ago (1996), the Orchid Recovery Program was established at Illinois College – a private liberal arts college – to promote the conservation of rare orchids through propagation. The school’s rural loca- tion, small size (1,000+ undergraduate students), and heavy teaching load (= 12 hours/semester) posed seri- ous challenges to the establishment of this research program. Nevertheless, over the years several aspects of the College were identified, targeted, and integrated to successfully blend the student academic experience with orchid conservation. A decade ago (1996), the Orchid Recovery Program was established at Illinois College – a private liberal arts college – to promote the conservation of rare orchids through propagation. The school’s rural loca- tion, small size (1,000+ undergraduate students), and heavy teaching load (= 12 hours/semester) posed seri- ous challenges to the establishment of this research program. Nevertheless, over the years several aspects of the College were identified, targeted, and integrated to successfully blend the student academic experience with orchid conservation.

An expanded role for in vitro symbiotic seed germination as a conservation tool: two case studies in North America (Platanthera leucophaea and Epidendrum nocturnum)

Interest in using mycorrhizal fungi to cultivate orchids from seed in vitro (=symbiotic seed germina- tion) has intensified in recent years and this approach is now an important conservation tool worldwide. In North America, symbiotic germination has been attempted for a growing number of orchid species in peril as a means to acquire seedlings suitable for reintroduction.Interest in using mycorrhizal fungi to cultivate orchids from seed in vitro (=symbiotic seed germina- tion) has intensified in recent years and this approach is now an important conservation tool worldwide. In North America, symbiotic germination has been attempted for a growing number of orchid species in peril as a means to acquire seedlings suitable for reintroduction

Symbiotic germination of three semi-aquatic rein orchids (Habenaria repens, H. quinquiseta, H. macroceratitis) from Florida

Abstract The destruction of wetlands in populated areas (e.g. Florida) has prompted interest in habitat restoration. We describe a symbiotic technique to germinate seeds of three semi-aquatic rein orchid species from Florida (Habenaria repens, H. quinquiseta, H. macroceratitis) and to cultivate H. repens seedlings on soil ex vitro. Seeds of all three Habenaria spp. germinated within 21 days of inoculation. Leaf-bearing seedlings of H. repens were obtained using two fungal isolates (Epulorhiza spp.) recovered from Florida orchids Spiranthes brevilabris and Epidendrum conopseum. Seedlings infected with the S. brevilabris fungus that were transferred to peat in a greenhouse had the highest (88.9%) survival (>159 days ex vitro). One H. repens seedling initiated anthesis 18 months after seed sowing. The methods outlined by this study have the potential to be adopted by wetland restoration projects seeking to include an orchid (H. repens) and mycorrhizal fungi as biotic agents

Crossing effects on seed viability and experimental germination of the federal threatened Platanthera leucophaea (Orchidaceae)

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In Vitro Seed Germination and Seedling Development of <i>Dracula felix</i> (Luer) Luer—An Orchid Native to Ecuador

Effects of daily temperature fluctuations that mimic on-site environmental conditions were tested on seed germination and development in Dracula felix, a native epiphytic orchid from the neotropics. Mature seeds collected from a native population lost their viability from 60% to 37.78% and 0% after 8 and 16 weeks., respectively, under 22 ± 2 °C. Seed viability was completely lost when seeds were maintained at −10 °C in the dark. Less than 50% germination was observed in D. felix seed across all treatments. Seed germinated regardless of the light or temperature treatment. However, significant improvement in germination was observed at 17/22 °C compared to constant temperature treatments. Early seedling development stages were observed only on 1/2XMS and VW media at 17 °C or 17 °C/22 °C under a 12 h light photoperiod. Neither germination nor seedling development were improved by any fungal strain tested using standard symbiotic germination protocols. Information obtained from this study is critical to ensure the ex-situ conservation of this and other rare Dracula species under current and future climate change scenarios