Royal Botanic Gardens Victoria
  • Melbourne, Victoria, Australia
Recent publications
Background Long-read sequencing platforms offered by Oxford Nanopore Technologies (ONT) allow native DNA containing epigenetic modifications to be directly sequenced, but can be limited by lower per-base accuracies. A key step post-sequencing is basecalling, the process of converting raw electrical signals produced by the sequencing device into nucleotide sequences. This is challenging as current basecallers are primarily based on mixtures of model species for training. Here we utilise both ONT PromethION and higher accuracy PacBio Sequel II HiFi sequencing on two plants, Phebalium stellatum and Xanthorrhoea johnsonii , to train species-specific basecaller models with the aim of improving per-base accuracy. We investigate sequencing accuracies achieved by ONT basecallers and assess accuracy gains by training single-species and species-specific basecaller models. We also evaluate accuracy gains from ONT’s improved flowcells (R10.4, FLO-PRO112) and sequencing kits (SQK-LSK112). For the truth dataset for both model training and accuracy assessment, we developed highly accurate, contiguous diploid reference genomes with PacBio Sequel II HiFi reads. Results Basecalling with ONT Guppy 5 and 6 super-accurate gave almost identical results, attaining read accuracies of 91.96% and 94.15%. Guppy’s plant-specific model gave highly mixed results, attaining read accuracies of 91.47% and 96.18%. Species-specific basecalling models improved read accuracy, attaining 93.24% and 95.16% read accuracies. R10.4 sequencing kits also improve sequencing accuracy, attaining read accuracies of 95.46% (super-accurate) and 96.87% (species-specific). Conclusions The use of a single mixed-species basecaller model, such as ONT Guppy super-accurate, may be reducing the accuracy of nanopore sequencing, due to conflicting genome biology within the training dataset and study species. Training of single-species and genome-specific basecaller models improves read accuracy. Studies that aim to do large-scale long-read genotyping would primarily benefit from training their own basecalling models. Such studies could use sequencing accuracy gains and improving bioinformatics tools to improve study outcomes.
Gleichenia (Gleicheniaceae) is a genus of distinctive branching ferns well represented in Australasia (Australia, New Caledonia, and New Zealand) where seven of the total eleven species occur. Published chloroplast phylogenies have shown that G. dicarpa is polyphyletic in New Zealand, and that one species from Madagascar and Réunion previously included in Gleichenia represented a separate newly-recognised genus, Rouxopteris. Here, chloroplast rbcL, trnL-trnF and combined trnL-trnF and rps4-trnS phylogenies are produced for all Australasian species. Australasian species formed a clade sister to the type of Gleichenia, G. polypodioides, confirming their placement in Gleichenia. Gleichenia dicarpa occurred in three major clades, which also contained other species or undescribed forms. The three separate lineages of G. dicarpa were morphologically distinct from these other species or forms; however, the three separate chloroplast lineages of G. dicarpa are difficult to morphologically distinguish from each other. Further study is required to elucidate whether the polyphyly of G. dicarpa could be due to unrecognised cryptic species, hybridisation, or incomplete lineage sorting. Low rbcL variation between all Australasian species suggests a radiation in the last five million years and at least six long-distance dispersal events in Australasia are inferred, comprising five between Australia and New Zealand, and one between Australia and New Caledonia.
Fungal taxonomy is a fundamental discipline that aims to recognise all fungi and their kinships. Approximately 5% of a practical estimate of 2.2–3.8 million species globally are currently known, and consequently the Fungal Tree of Life (FTOL) is very incompletely reconstructed. With the advances of new technologies, mycology is marching into the interdisciplinary and globalisation era. To make fungal taxonomy relevant, innovative sampling methods and phylogenomics analyses should be performed to reconstruct a much more comprehensive FTOL. In association with this densely sampled FTOL, multiomics will reveal what drives fungal species diversification and how fungal traits evolve to adapt to various environments, while metagenomics will facilitate the understanding and protection of the ecological functions of fungi. A coordinated approach to pursuing these research agendas that includes conceiving of and costing a mission to describe all the fungi on the planet will unlock potential of fungi to support sustainable development of our society.
The consumption of fungi by animals is a significant trophic interaction in most terrestrial ecosystems, yet the role mammals play in these associations has been incompletely studied. In this review, we compile 1 154 references published over the last 146 years and provide the first comprehensive global review of mammal species known to eat fungi (508 species in 15 orders). We review experimental studies that found viable fungal inoculum in the scats of at least 40 mammal species, including spores from at least 58 mycorrhizal fungal species that remained viable after ingestion by mammals. We provide a summary of mammal behaviours relating to the consumption of fungi, the nutritional importance of fungi for mammals, and the role of mammals in fungal spore dispersal. We also provide evidence to suggest that the morphological evolution of sequestrate fungal sporocarps (fruiting bodies) has likely been driven in part by the dispersal advantages provided by mammals. Finally, we demonstrate how these interconnected associations are widespread globally and have far-reaching ecological implications for mammals, fungi and associated plants in most terrestrial ecosystems.
Purpose Owing to their topographic location and nutrient rich soils, riparian forests are often converted to pastures for grazing. In recent decades, remnant riparian forests cleared for grazing pastures have been restored with native species. The impacts of such land-use changes on soil fungal communities are unclear, despite the central roles that soil fungi play in key ecosystem processes. We investigated how soil fungal taxonomic and functional composition are affected by land-use change at different depths, and if variation in soil fungal communities is related to edaphic properties and extant vegetation. Methods The study was conducted in six waterways in south-eastern Australia, each comprising three land-use types: remnant riparian forest, cleared forest converted to pasture, and pastures restored with native plants. We surveyed three strata of vegetation and sampled top-soil and sub-soil to characterise physicochemical properties and soil fungal communities. ITS1 region sequences were used to assign soil fungal taxonomic and functional composition. Results Fungal taxonomic and functional composition infrequently varied with land-use change or soil depth. Overall, environmental properties (soil and vegetation) explained 35–36% of variation in both fungal taxonomic and functional composition. Soil fungal taxonomic composition was related to soil fertility (N, P, K, pH and Ca) and ground cover characteristics, whereas functional composition was related to clay content, sub-canopy cover and tree basal area. Conclusion Across the six studied waterways, fungal taxonomic and functional composition were more strongly associated with land-use mediated changes in site-scale soil physicochemical properties and vegetation structure than broad-scale classes of land-use type.
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. We investigated the mycorrhizal associations of five Australian Cryptostylis species (27 sites sampled) and included limited sampling from three Asiatic Cryptostylis species (two sites), using fungal isolation and molecular approaches. 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.
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.
International trade in plants and climate change are two of the main factors causing damaging tree pests (i.e. fungi and insects) to spread into new areas. To mitigate these risks, a large-scale assessment of tree-associated fungi and insects is needed. We present records of endophytic fungi and insects in twigs of 17 angiosperm and gymnosperm genera, from 51 locations in 32 countries worldwide. Endophytic fungi were characterized by high-throughput sequencing of 352 samples from 145 tree species in 28 countries. Insects were reared from 227 samples of 109 tree species in 18 countries and sorted into taxonomic orders and feeding guilds. Herbivorous insects were grouped into morphospecies and were identified using molecular and morphological approaches. This dataset reveals the diversity of tree-associated taxa, as it contains 12,721 fungal Amplicon Sequence Variants and 208 herbivorous insect morphospecies, sampled across broad geographic and climatic gradients and for many tree species. This dataset will facilitate applied and fundamental studies on the distribution of fungal endophytes and insects in trees.
The use of direct seeding for revegetation often results in poor recruitment outcomes. For many species, it is unclear where recruitment bottlenecks occur in the transitions between early life-history stages and how soil moisture conditions affect these bottlenecks. Thus, we asked: (1) which life-history stage transitions are most limiting to seedling recruitment? and (2) how do soil moisture levels affect recruitment? Using a field-based trial, we quantified the recruitment process from a seed to seedling for two woody Acacia species. Using a novel technique, in which seeds were confined to germination caches (small baskets), along with the use of germination bags, we assessed pre-emergence processes at regular intervals. Seeds sown directly into the soil in adjacent rows were intensively monitored to assess post-emergence processes. To investigate the effects of soil moisture on seedling recruitment, a glasshouse experiment assessed transitions between life-history stages under three different soil moisture treatments. In the field, the transition between a seed and germinated seed limited recruitment more than all other life-history stage transitions combined, with 32%–50% of seeds not germinating for the two species. Approximately one third of seeds of both species died prior to germinating, with few seeds remaining dormant. In the glasshouse trial, seed germination increased with increasing soil moisture, however, so did the extent of seed death. Our results suggest that the transition from a seed to germinated seed was the most limiting bottleneck to recruitment, mostly due to seed mortality rather than dormancy processes, with pathogen attack the most likely cause. As increased soil moisture both promoted germination but also seed mortality, understanding the optimal soil moisture thresholds that maximise germination and the transition to an established plant is essential in maximising direct seeding outcomes. Identifying the life-history stage transitions most limiting to plant recruitment may allow management to target specific bottlenecks in order to improve direct seeding outcomes.
Climate change and other global change drivers threaten plant diversity in mountains worldwide. A widely documented response to such environmental modifications is for plant species to change their elevational ranges. Range shifts are often idiosyncratic and difficult to generalize, partly due to variation in sampling methods. There is thus a need for a standardized monitoring strategy that can be applied across mountain regions to assess distribution changes and community turnover of native and non‐native plant species over space and time. Here, we present a conceptually intuitive and standardized protocol developed by the Mountain Invasion Research Network (MIREN) to systematically quantify global patterns of native and non‐native species distributions along elevation gradients and shifts arising from interactive effects of climate change and human disturbance. Usually repeated every five years, surveys consist of 20 sample sites located at equal elevation increments along three replicate roads per sampling region. At each site, three plots extend from the side of a mountain road into surrounding natural vegetation. The protocol has been successfully used in 18 regions worldwide from 2007 to present. Analyses of one point in time already generated some salient results, and revealed region‐specific elevational patterns of native plant species richness, but a globally consistent elevational decline in non‐native species richness. Non‐native plants were also more abundant directly adjacent to road edges, suggesting that disturbed roadsides serve as a vector for invasions into mountains. From the upcoming analyses of time series, even more exciting results can be expected, especially about range shifts. Implementing the protocol in more mountain regions globally would help to generate a more complete picture of how global change alters species distributions. This would inform conservation policy in mountain ecosystems, where some conservation policies remain poorly implemented. We summarize the findings achieved with the standardized sampling protocol developed by the Mountain Invasion Research Network (MIREN) for monitoring the impact of global change on elevational plant species distributions. We intend to promote the use of the protocol to generate global insights into native and non‐native species responses to rapid global change in mountains.
Auriculariales accommodates species with diverse basidiomes and hymenophores. From morphological and phylogenetic perspectives, we perform a taxonomic study on Heteroradulum , a recently validated genus within the Auriculariales . The genus Grammatus is merged into Heteroradulum , and thus its generic type G. labyrinthinus is combined with Heteroradulum and G. semis is reaccepted as a member of Heteroradulum . Heteroradulum australiense is newly described on the basis of three Australian specimens. Heteroradulum yunnanense is excluded from this genus and its taxonomic position at the generic level is considered uncertain. Accordingly, the circumscription of Heteroradulum is re-delimited and the concept of this genus is adjusted by including irpicoid to poroid hymenophores and a hyphal system with clamp connections or simple septa. A key to all nine accepted species of Heteroradulum is presented.
Reduced precipitation in the Miocene triggered the geographic contraction of rainforest ecosystems around the world. In Australia, this change was particularly pronounced; mesic rainforest ecosystems that once dominated the landscape transformed into the shrublands, grasslands, and deserts of today. A lack of well-preserved fossils has made it difficult to understand the nature of Australian ecosystems before the aridification. Here, we report on an exceptionally well-preserved rainforest biota from New South Wales, Australia. This Konservat-Lagerstätte hosts a rich diversity of microfossils, plants, insects, spiders, and vertebrate remains preserved in goethite. We document evidence for several species interactions including predation, parasitism, and pollination. The fossils are indicative of an oxbow lake in a mesic rainforest and suggest that rainforest distributions have shifted since the Miocene. The variety of fossils preserved, together with high fidelity of preservation, allows for unprecedented insights into the mesic ecosystems that dominated Australia during the Miocene.
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a particularly useful tool to determine the localization of metabolites in plant tissues enabling the exploration of spatial metabolic processes in plant biology. In this case study, we examined cyanogenic glycosides (CNglycs), in the delicate floral tissues of highly cyanogenic Grevillea robusta (southern silky oak) and Macadamia tetraphylla (macadamia nut) collected in the field. CNglycs are canonical defence metabolites, but more recent work indicates they may play a much broader role in plant metabolism. We quantified CNglycs in floral tissues by chemical assay and visualized their spatial distribution using MALDI-MSI to explore their biochemical and ecological roles. The species examined contained two tyrosine-derived CNglycs, the monoglycoside dhurrin, and diglycoside proteacin, identified using HP-TLC and LC-MS/MS analyses. Within florets, the most cyanogenic tissue was the style for both species. MALDI-MSI revealed different distributions of dhurrin and proteacin within florets of both species and differences in the ratios of dhurrin to proteacin between specific floral tissues. Dhurrin tended to be mostly localized in epidermal layers, consistent with a role in defence, while proteacin was distributed more evenly throughout other floral tissues indicating other potential roles.
Tylocinum Y.C. Li & Zhu L. Yang 2016 is a Boletaceae genus belonging in subfamily Leccinoideae. It was described in 2016 from China and, prior to this study, it contained only one species, T. griseolum Y.C. Li & Zhu L. Yang 2016. During our survey of Boletaceae from Thailand, we collected some specimens that could be identified as a Tylocinum species, different from T. griseolum . The bolete specimens, collected in forests dominated by Dipterocarpaceae and Fagaceae in northern Thailand, are described as Tylocinum brevisporum Raghoonundon & Raspé sp. nov. Macroscopic and microscopic descriptions with illustrations are provided, as well as a 3-gene phylogeny, which confirms the new taxon’s position in Tylocinum . Tylocinum brevisporum differs from the only other known Tylocinum species ( T. griseolum) by its brownish-grey colour, greyish-orange to brownish-orange colour change in the hymenophore when bruised, smaller pores (≤ 0.5 mm), longer tubes (up to 6 mm long), shorter and narrower basidiospores, longer and broader basidia and longer pleurocystidia relative to cheilocystidia. T. brevisporum is the second species from the genus Tylocinum and the only one to be found outside China thus far.
Organelle genomes are typically represented as single, static, circular molecules. However, there is evidence that the chloroplast genome exists in two structural haplotypes and that the mitochondrial genome can display multiple circular, linear or branching forms. We sequenced and assembled chloroplast and mitochondrial genomes of the Golden Wattle, Acacia pycnantha, using long reads, iterative baiting to extract organelle-only reads, and several assembly algorithms to explore genomic structure. Using a de novo assembly approach agnostic to previous hypotheses about structure, we found that different assemblies revealed contrasting arrangements of genomic segments; a hypothesis supported by mapped reads spanning alternate paths.
Three Australian species with sequestrate basidiome forms are recorded for the first time in the genus Lactifluus based on nuclear ITS-LSU and morphological data. These species represent three rare independent evolutionary events resulting in sequestrate basidiomes arising from agaricoid species in three different sections in two subgenera. All three species have highly reduced basidiome forms, and no species with intermediate forms have been found. Lactifluus dendriticus is unique in the genus in having highly branched, dendritic terminal elements in the pileipellis. We provide full descriptions of two species: Zelleromyces dendriticus (= Lactifluus dendriticus comb. nov. ) in Lactifluus subg. Lactifluus sect. Gerardii , and Lactifluus geoprofluens sp. nov. in Lf . subg. Lactifluus sect. Lactifluus . A reduced description is provided for the third, Lactifluus sp. prov. KV181 in Lf . subg. Pseudogymnocarpi sect. Pseudogymnocarpi , as it is currently known from a single sequence.
Hysterangiales ( Phallomycetidae , Agaricomycetes , Basidiomycota ) is a diverse, nearly cosmopolitan order of predominantly hypogeous, sequestrate, ectomycorrhizal fungi. Expanding on previously published phylogenies, we significantly increased sampling of Hysterangiales specimens, emphasizing representatives from Australia. Using protein-coding genes atp6 (adenosine triphosphate synthase subunit 6) and tef1 (translation elongation factor 1-α), we recovered 26 provisional novel genera, and corroborated existing genera and families. Further, two new suborders ( Phallogastrineae subord. nov. and Hysterangineae subord. nov. ) and a new family ( Phallogastraceae fam. nov. ) are described, and three new combinations made to Phallogaster . Aspects of classification and biogeography are presented.
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