The Role of Photobionts as Drivers of Diversification in an Island Radiation of Lichen-Forming Fungi

Speciation in oceanic islands has attracted the interest of scientists since the 19th century. One of the most striking evolutionary phenomena that can be studied in islands is adaptive radiation, that is, when a lineage gives rise to different species by means of ecological speciation. Some of the best-known examples of adaptive radiation are charismatic organisms like the Darwin finches of the Galapagos and the cichlid fishes of the great African lakes. In these and many other examples, a segregation of the trophic niche has been shown to be an important diversification driver. Radiations are known in other groups of organisms, such as lichen-forming fungi. However, very few studies have investigated their adaptive nature, and none have focused on the trophic niche. In this study, we explore the role of the trophic niche in a putative radiation of endemic species from the Macaronesian Region, the Ramalina decipiens group. The photobiont diversity was studied by Illumina MiSeq sequencing of the ITS2 region of 197 specimens spanning the phylogenetic breadth and geographic range of the group. A total of 66 amplicon sequence variants belonging to the four main clades of the algal genus Trebouxia were found. Approximately half of the examined thalli showed algal coexistence, but in most of them, a single main photobiont amounted to more than 90% of the reads. However, there were no significant differences in photobiont identity and in the abundance of ITS2 reads across the species of the group. We conclude that a segregation of the trophic niche has not occurred in the R. decipiens radiation.

Variation in Fungal Community in Grapevine (Vitis vinifera) Nursery Stock Depends on Nursery, Variety and Rootstock

Grapevine trunk diseases (GTDs) are caused by cryptic complexes of fungal pathogens and have become a growing problem for new grapevine (Vitis vinifera) plantations. We studied the role of the nursery, variety, and rootstock in the composition of the fungal communities in root collars and graft unions of planting material in Catalonia (NE Spain). We compared necrosis and fungal communities in graft unions and root collars at harvest, and then after three months of cold storage. We evaluated combinations of eleven red and five white varieties with four common rootstocks coming from six nurseries. Fungal communities were characterized by isolation and metabarcoding of the ITS2 region. Our data suggests that nursery followed by rootstock and variety had significant effects on necrosis and fungal community structure in graft and root tissues. Within the plant, we found large differences in terms fungal community distribution between graft and root tissues. Graft unions housed a significantly higher relative abundance of GTD-related Operational Taxonomic Units (OTUs) than root collars. More severe necrosis was correlated with a lower relative abundance of GTD-related OTUs based on isolation and metabarcoding analyses. Our results suggest that nurseries and therefore their plant production practices play a major role in determining the fungal and GTD-related fungal community in grapevine plants sold for planting. GTD variation across rootstocks and varieties could be explored as a venue for minimizing pathogen load in young plantations.

DNA barcoding of Naididae (Annelida, Oligochaeta), based on cytochrome C oxidase gene and ITS2 region in China

Exploring the effectiveness of DNA barcoding in species identification is a prerequisite for biodiversity conservation and environmental monitoring. Aquatic oligochaetes could serve as excellent indicators in aquatic monitoring programmes. However, few studies have examined the effectiveness of DNA barcoding in these specific organisms. The mitochondrial cytochrome C oxidase (COI) gene of 83 specimens belonging to 40 species of 18 genera were sequenced in this study. The results showed that there was a barcode gap between species of Naididae and the intraspecific genetic distances of each species were smaller than interspecific genetic distances. The classification results of ABGD (Automatic Barcode Gap Discovery) were consistent with those of morphological identification, except for Tubifex tubifex and Lumbriculus variegatus. All species were successfully distinguished in the phylogenetic tree, based on the ITS2 region, which was coincident with the morphological result. Our results provided evidence that DNA barcoding can be used as an effective and convenient tool for species identification of the family Naididae and even for other aquatic oligochaetes.

Aquatic Hyphomycete Taxonomic Relatedness Translates into Lower Genetic Divergence of the Nitrate Reductase Gene

Aquatic hyphomycetes are key microbial decomposers in freshwater that are capable of producing extracellular enzymes targeting complex molecules of leaf litter, thus, being crucial to nutrient cycling in these ecosystems. These fungi are also able to assimilate nutrients (e.g., nitrogen) from stream water, immobilizing these nutrients in the decomposing leaf litter and increasing its nutritional value for higher trophic levels. Evaluating the aquatic hyphomycete functional genetic diversity is, thus, pivotal to understanding the potential impacts of biodiversity loss on nutrient cycling in freshwater. In this work, the inter- and intraspecific taxonomic (ITS1-5.8S-ITS2 region) and functional (nitrate reductase gene) diversity of 40 aquatic hyphomycete strains, belonging to 23 species, was evaluated. A positive correlation was found between the taxonomic and nitrate reductase gene divergences. Interestingly, some cases challenged this trend: Dactylella cylindrospora (Orbiliomycetes) and Thelonectria rubi (Sordariomycetes), which were phylogenetically identical but highly divergent regarding the nitrate reductase gene; and Collembolispora barbata (incertae sedis) and Tetracladium apiense (Leotiomycetes), which exhibited moderate taxonomic divergence but no divergence in the nitrate reductase gene. Additionally, Tricladium chaetocladium (Leotiomycetes) strains were phylogenetically identical but displayed a degree of nitrate reductase gene divergence above the average for the interspecific level. Overall, both inter- and intraspecific functional diversity were observed among aquatic hyphomycetes.

Metabarcoding of Soil Fungal Communities from Perturbed Areas Reveal Drive Role of Environmental Factors in Microbial Diversity of Colombian Andosols

Changes in soil fungal community caused by land use have not been sufficiently studied in South-American Andosols, considered globally as important food production areas. This study analyzed 26 soil samples of Andosols collected from locations devoted to conservation, agriculture and mining activities in the southeastern region of Antioquia, Colombia, to establish differences between fungal communities as indicators of the degree of soil perturbation. The study developed a novel heminested PCR with primers SSUmCf Mix, ITS4 and fITS7 to assess Arbuscular Mycorrhizal Fungi detection in a Illumina MiSeq metabarcoding on nuclear ribosomal ITS2 region. A non-metric multidimensional scaling allowed exploring driver factors of fungal community changes, while fitted Dirichlet-multinomial models and PERMANOVA tests allowed identifying the correlations between alpha diversity indexes and community dissimilarities, as well as the significance of land use effects on fungal community composition. Furthermore, response ratios were determined to assess effect size by land use over relevant taxa. Results suggest a good coverage of fungal diversity with a detection of 10,529 high-quality ITS2 sequences belonged to phylum Glomeromycota. The analysis shows strong correlations of Shannon and Fisher indexes with dissimilarities on fungal communities among land uses (r=0.94), related to variations in temperature, air humidity and organic matter contents that lead to significant responses in abundances of relevant orders (such as Wallemiales and Trichosporonales). The study highlights the rich fungal biodiversity of the tropical Andosols, their specific sensitivities to environmental perturbation factors, and the useful range of a metabarcoding approach to characterize soil fungal communities.

Symbiodinium diversity and potential hybridisation on the highly biodiverse coral reefs of Timor-Leste

<p>To persist in oligotrophic waters, reef-building corals rely on nutritional interactions with their intracellular symbionts: photosynthetic dinoflagellates of the genus Symbiodinium. This relationship is threatened by increasing environmental stress, which can stimulate loss of these symbionts from coral tissues (‘coral bleaching’). Members of the genus Symbiodinium display high levels of genetic diversity, and demonstrate a corresponding diversity in physiological responses to environmental change. However, the true diversity and potential for genetic adaptation in this genus remain poorly characterised. This thesis aimed to further the understanding of symbiont diversity and adaptive potential by conducting assessments of Symbiodinium at Atauro Island and the neighbouring Timor-Leste mainland. These sites have previously been shown to be of outstanding conservation value, with extremely high levels of coral diversity. Atauro Island also possibly hosts the highest diversity of reef fish in the world. However, the Symbiodinium communities at these sites have never been assessed. Two specific objectives were therefore addressed here. The first was to measure Symbiodinium diversity at Atauro Island (four sites) and Timor (three sites), using direct sequencing of three gene regions: cob gene, mitochondrion; ITS2 region, nucleus; and psbAncr region, chloroplast; in addition to Next Generation Sequencing of the ITS2 region. The second objective was to establish evidence for Symbiodinium hybridisation, a potentially rapid evolutionary mechanism that may facilitate adaptation to environmental stress, by looking for genetic incongruences between Symbiodinium organelles. Coral symbionts of Timor-Leste were found to be similar to those from other coral reefs of the Indo-Pacific, with several host generalist and multiple host specific types observed. However, there were also several novel Symbiodinium types found (C15p, C15q, C1x, C1z). Despite their geographic proximity, there were strong differences observed between the symbiont communities of Atauro Island and mainland Timor. In particular, the coral genus Pocillopora hosted clade C symbionts exclusively at Atauro Island, while it hosted clade D exclusively at Timor sites. Other symbiont types also showed geographic partitioning, and diversity was 1.25 times higher at Atauro Island, a figure consistent for the cob and ITS2 regions. While Timor sites have comparable Symbiodinium diversity to other reefs globally, Atauro has noticeably elevated Symbiodinium diversity. Next Generation Sequencing affirmed these patterns, with Atauro Island sites having much more diverse cryptic populations of Symbiodinium, largely driven by symbionts in clade C. The exception was clade D symbionts, which were proportionally far more diverse at Timor, a pattern consistent in multiple coral genera. There was strong evidence of genetic incongruence at two Atauro Island sites, with all testing procedures identifying genetic discordance between organellar and nuclear genomes, consistent with theoretical predictions of hybridisation. This study therefore presents strong evidence for Symbiodinium hybridisation, and its corroboration by multiple loci is significant. Putative hybrid Symbiodinium always had a common type as one of the possible parents, with a rarer symbiont as the other. For example, one putative hybrid had organellar genes of the common generalist Symbiodinium C40, while it was identified as the rare type C3z with the ITS2 region. Both of these Symbiodinium types were also found in congruent relationships, which strongly supports the possibility that they sexually reproduced to produce the incongruent putative hybrid. Environmental stressors, such as increased temperature, turbidity and sedimentation, are suggested reasons for lowered Symbiodinium diversity at Timor, as they may impose a selection pressure on corals to only keep highly beneficial symbionts. This reduction in diversity likely limits the potential for adaptive change through methods like hybridisation, and highlights the need to assess and conserve symbiont diversity to the same extent as coral diversity.</p>

Symbiodinium diversity and potential hybridisation on the highly biodiverse coral reefs of Timor-Leste

<p>To persist in oligotrophic waters, reef-building corals rely on nutritional interactions with their intracellular symbionts: photosynthetic dinoflagellates of the genus Symbiodinium. This relationship is threatened by increasing environmental stress, which can stimulate loss of these symbionts from coral tissues (‘coral bleaching’). Members of the genus Symbiodinium display high levels of genetic diversity, and demonstrate a corresponding diversity in physiological responses to environmental change. However, the true diversity and potential for genetic adaptation in this genus remain poorly characterised. This thesis aimed to further the understanding of symbiont diversity and adaptive potential by conducting assessments of Symbiodinium at Atauro Island and the neighbouring Timor-Leste mainland. These sites have previously been shown to be of outstanding conservation value, with extremely high levels of coral diversity. Atauro Island also possibly hosts the highest diversity of reef fish in the world. However, the Symbiodinium communities at these sites have never been assessed. Two specific objectives were therefore addressed here. The first was to measure Symbiodinium diversity at Atauro Island (four sites) and Timor (three sites), using direct sequencing of three gene regions: cob gene, mitochondrion; ITS2 region, nucleus; and psbAncr region, chloroplast; in addition to Next Generation Sequencing of the ITS2 region. The second objective was to establish evidence for Symbiodinium hybridisation, a potentially rapid evolutionary mechanism that may facilitate adaptation to environmental stress, by looking for genetic incongruences between Symbiodinium organelles. Coral symbionts of Timor-Leste were found to be similar to those from other coral reefs of the Indo-Pacific, with several host generalist and multiple host specific types observed. However, there were also several novel Symbiodinium types found (C15p, C15q, C1x, C1z). Despite their geographic proximity, there were strong differences observed between the symbiont communities of Atauro Island and mainland Timor. In particular, the coral genus Pocillopora hosted clade C symbionts exclusively at Atauro Island, while it hosted clade D exclusively at Timor sites. Other symbiont types also showed geographic partitioning, and diversity was 1.25 times higher at Atauro Island, a figure consistent for the cob and ITS2 regions. While Timor sites have comparable Symbiodinium diversity to other reefs globally, Atauro has noticeably elevated Symbiodinium diversity. Next Generation Sequencing affirmed these patterns, with Atauro Island sites having much more diverse cryptic populations of Symbiodinium, largely driven by symbionts in clade C. The exception was clade D symbionts, which were proportionally far more diverse at Timor, a pattern consistent in multiple coral genera. There was strong evidence of genetic incongruence at two Atauro Island sites, with all testing procedures identifying genetic discordance between organellar and nuclear genomes, consistent with theoretical predictions of hybridisation. This study therefore presents strong evidence for Symbiodinium hybridisation, and its corroboration by multiple loci is significant. Putative hybrid Symbiodinium always had a common type as one of the possible parents, with a rarer symbiont as the other. For example, one putative hybrid had organellar genes of the common generalist Symbiodinium C40, while it was identified as the rare type C3z with the ITS2 region. Both of these Symbiodinium types were also found in congruent relationships, which strongly supports the possibility that they sexually reproduced to produce the incongruent putative hybrid. Environmental stressors, such as increased temperature, turbidity and sedimentation, are suggested reasons for lowered Symbiodinium diversity at Timor, as they may impose a selection pressure on corals to only keep highly beneficial symbionts. This reduction in diversity likely limits the potential for adaptive change through methods like hybridisation, and highlights the need to assess and conserve symbiont diversity to the same extent as coral diversity.</p>

Mycorrhizal Fungal Partners Remain Constant during a Root Lifecycle of Pleione bulbocodioides (Orchidaceae)

Mycorrhizal mutualisms are vital for orchids through germination to adulthood. Fungal species diversity and community composition vary across seasons and plant development stages and affect plant survival, adaptation, and community maintenance. Knowledge of the temporal turnover of mycorrhizal fungi (OMF) remains poorly understood in the eco-physiologically diverse orchids (especially in epiphytic orchids), although it is important to understand the function and adaptation of mycorrhizae. Some species of Pleione are epiphytic plants with annual roots and may recruit different fungal partners during their root lifecycle. Based on continuous samplings of Pleione bulbocodioides during a whole root lifecycle, we characterized the fungal temporal dynamics using Illumina sequencing of the ITS2 region. Our data showed that the plants of P. bulbocodioides were quickly colonized by OMF at root emergence and had a constant OMF composition throughout one root lifecycle, although the OMF richness declined with root aging after a peak occurrence during root elongation. In contrast, the richness of root-inhabiting fungal endophytes kept increasing with root aging and more drastic turnovers were found in their species compositions. Our findings of OMF temporal turnover contribute to further understanding of mycorrhizal associations and adaptation of Orchidaceae and will benefit orchid resource conservation and utilization.

Distribution and diversity of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) in a South African nature reserve

Entomopathogenic nematodes (EPNs) are microscopic roundworms that are found in soil worldwide. They deliver an important ecosystem service through preventing natural flares in insect reproduction by means of utilising the soil stages of insects as a food source and by acting as natural biocontrol agents. A survey of EPNs was conducted in the JS Marais Nature Reserve, Stellenbosch, in the Western Cape province of South Africa. Soil samples were baited with the larvae of three susceptible hosts, codling moth (Cydia pomonella), wax moth (Galleria mellonella) and mealworm (Tenebrio molitor) to determine the presence of EPN. Of the 76 soil samples collected across the reserve, 39 were found to be positive for the presence of EPN (51.32%). Among the positive samples, 87% contained Steinernema isolates, 8% contained Heterorhabditis and 5% contained the Oscheius sp. Morphological and molecular studies were performed to characterise the isolates to species level. The Steinernema species were identified as Steinernema khoisanae in 34 samples, and as Steinernema nguyeni in five samples. The only species of Heterorhabditis found was H. safricana, which was identified from three samples. An unknown Oscheius sp. was found in two samples. The reserve’s population of S. khoisanae showed interesting inter-individual variation (93%) early in the internal transcribe spacer (ITS) region, leading to short single-usable sequences, which, in most cases, included only the ITS1 or ITS2 region. However, using the D2D3 confirmed their identity as S. khoisanae, with such occurring in all areas and soil types of the reserve.Conservation implications: The undisturbed alluvial fynbos and renosterveld of the JS Marais Nature Reserve showed high EPN abundance and diversity in stark contrast to the agro-ecosystems present in the Cape floristic region. This finding, on a micro level, should be conserved for future bioprospecting in the fynbos for EPNs with potential as biocontrol agents.

Metabolomics-Driven Discovery of an Introduced Species and Two Malaysian Piper betle L. Variants

The differences in pungency of “sirih” imply the probable occurrence of several variants of Piper betle L. in Malaysia. However, the metabolite profiles underlying the pungency of the different variants remain a subject of further research. The differences in metabolite profiles of selected Malaysian P. betle variants were thus investigated; specifically, the leaf aqueous methanolic extracts and essential oils were analyzed via 1H-NMR and GC-MS metabolomics, respectively. Principal component analysis (PCA) of the 1H-NMR spectral data showed quantitative differences in the metabolite profiles of “sirih melayu” and “sirih india” and revealed an ambiguous group of samples with low acetic acid content, which was identified as Piper rubro-venosum hort. ex Rodigas based on DNA sequences of the internal transcribed spacer 2 (ITS2) region. The finding was supported by PCA of two GC-MS datasets of P. betle samples obtained from several states in Peninsular Malaysia, which displayed clustering of the samples into “sirih melayu” and “sirih india” groups. Higher abundance of chavicol acetate was consistently found to be characteristic of “sirih melayu”. The present research has provided preliminary evidence supporting the notion of occurrence of two P. betle variants in Malaysia based on chemical profiles, which may be related to the different genders of P. betle.