Strong phylogenetic congruence between Tulasnella fungi and their associated Drakaeinae orchids.

The study of congruency between phylogenies of interacting species can provide a powerful approach for understanding the evolutionary history of symbiotic associations. Orchid mycorrhizal fungi can survive independently of orchids making cospeciation unlikely, leading us to predict that any congruence would arise from host-switches to closely related fungal species. The Australasian orchid subtribe Drakaeinae is an iconic group of sexually deceptive orchids that consists of approximately 66 species. In this study, we investigated the evolutionary relationships between representatives of all six Drakaeinae orchid genera (39 species) and their mycorrhizal fungi. We used an exome capture dataset to generate the first well-resolved phylogeny of the Drakaeinae genera. A total of 10 closely related Tulasnella Operational Taxonomic Units (OTUs) and previously described species were associated with the Drakaeinae orchids. Three of them were shared among orchid genera, with each genus associating with 1-6 Tulasnella lineages. Cophylogenetic analyses show Drakaeinae orchids and their Tulasnella associates exhibit significant congruence (p < 0.001) in the topology of their phylogenetic trees. An event-based method also revealed significant congruence in Drakaeinae-Tulasnella relationships, with duplications (35), losses (25), and failure to diverge (9) the most frequent events, with minimal evidence for cospeciation (1) and host-switches (2). The high number of duplications suggests that the orchids speciate independently from the fungi, and the fungal species association of the ancestral orchid species is typically maintained in the daughter species. For the Drakaeinae-Tulasnella interaction, a pattern of phylogenetic niche conservatism rather than coevolution likely explains the observed phylogenetic congruency in orchid and fungal phylogenies. Given that many orchid genera are characterized by sharing of fungal species between closely related orchid species, we predict that these findings may apply to a wide range of orchid lineages.

Cryptostylis species (Orchidaceae) from a broad geographic and habitat range associate with a phylogenetically narrow lineage of Tulasnellaceae fungi.

While many Australian terrestrial orchids have highly specialized mycorrhizal associations, we tested the hypothesis that the geographically widespread orchid genus Cryptostylis associates with a diversity of fungal species. Using fungal isolation and molecular approaches, we investigated the mycorrhizal associations of five Australian Cryptostylis species (27 sites sampled) and included limited sampling from three Asiatic Cryptostylis species (two sites). Like related orchid genera, Tulasnellaceae formed the main fungal associations of the Cryptostylis species we sampled, although some ectomycorrhizal, ericoid and saprotrophic fungi were detected infrequently. Each species of Australian Cryptostylis associated with three to seven Tulasnella Operational Taxonomic Units (OTUs), except for C. hunteriana where only one Tulasnella OTU was detected. In total, eleven Tulasnella OTUs associated with Australian Cryptostylis. The Asiatic Cryptostylis associated with four different Tulasnella OTUs belonging to the same lineage as the Australian species. While five Tulasnella OTUs (T. australiensis, T. prima, T. warcupii, T. densa, and T. punctata) were used by multiple species of Australian Cryptostylis, the most commonly used OTU differed between orchid species. The association with different Tulasnella fungi by Cryptostylis species co-occurring at the same site suggests that in any given environmental condition, Cryptostylis species may intrinsically favour different fungal OTUs.

New species of Tulasnella associated with Australian terrestrial orchids in the subtribes Megastylidinae and Thelymitrinae.

Tulasnella (Tulasnellaceae) is a genus of fungus that can form mycorrhizal associations with orchids (Orchidaceae). Here we used molecular phylogenetic analyses and morphological characteristics of pure cultures across four different media to support the description of five new Tulasnella species associated with commonly occurring and endangered Australian orchids. Tulasnella nerrigaensis associates with Calochilus; T. subasymmetrica and T. kiataensis with Thelymitra; and T. korungensis and T. multinucleata with Pyrorchis and Rimacola respectively. The newly described species were primarily delimited by analyses of five loci: nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), C14436 (adenosine triphosphate [ATP] synthase), C4102 (glutamate synthase), C3304 (ATP helicase), and mt large subunit 16S rDNA (mtLSU). Tulasnella subasymmetrica is introduced for some isolates previously identified as T. asymmetrica, and this latter species is characterized from multilocus sequencing of a new isolate that matches ITS sequences from the ex-type culture. Morphological differences between the new species are slight. Tulasnella multinucleata has 6-12 nuclei per hyphal compartment which is the first instance of multinucleate rather than binucleate or trinucleate hyphal compartments in Tulasnella. The formal description of these species of Tulasnella will aid in future evolutionary and ecological studies of orchid-fungal interactions.

New species of Tulasnella associated with Australian terrestrial orchids in the Cryptostylidinae and Drakaeinae.

Many orchids have an obligate relationship with Tulasnella mycorrhizal fungi for seed germination and support into adulthood. Despite the importance of Tulasnella as mycorrhizal partners, many species remain undescribed. Here, we use multiple sequence locus phylogenetic analyses to delimit and describe six new Tulasnella species associated with Australian terrestrial orchids from the subtribes Cryptostylidinae and Drakaeinae. Five of the new species, Tulasnella australiensis, T. occidentalis, T. punctata, T. densa, and T. concentrica, all associate with Cryptostylis (Cryptostylidinae), whereas T. rosea associates with Spiculaea ciliata (Drakaeinae). Isolates representing T. australiensis were previously also reported in association with Arthrochilus (Drakaeinae). All newly described Tulasnella species were delimited by phylogenetic analyses of four loci (nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 [ITS], C14436 [ATP synthase], C4102 [glutamate synthase], and mt 16S rDNA [mtLSU]). The pairwise sequence divergence between species for the ITS region ranged from 5.6% to 25.2%, and the maximum sequence divergence within the newly described species ranged from 1.64% to 4.97%. There was a gap in the distribution of within- and between-species pairwise divergences in the region of 4-6%, with only one within-species value of 4.97% (for two T. australiensis isolates) and one between-species value of 5.6% (involving an isolate of T. occidentalis) falling within this region. Based on fluorescence staining, all six new Tulasnella species are binucleate and have septate, cylindrical hyphae. There was some subtle variation in culture morphology, but colony diameter as measured on 3MN+vitamin medium after 6 wk of growth did not differ among species. However, T. australiensis grew significantly (P < 0.02) slower than others on ½ FIM and » potato dextrose agar (PDA) media. Formal description of these Tulasnella species contributes significantly to documentation of Tulasnella diversity and provides names and delimitations to underpin further research on the fungi and their relationships with orchids.