Hooghännaliste (Collembola) ja nendega seotud seeneliikide molekulaarne määramine

Väitekirja elektrooniline versioon ei sisalda publikatsiooneMuld on mitmekesine elupaik, mis hõlmab suurt mikroobide ja loomade liigirikkust. Rikkalik mullaelustik on olulisel kohal paljudes looduslikes protsessides alates mulla kujundamisest ja lagundamisprotsessidest kuni mikrokliima reguleerimiseni. Molekulaarsete identifitseerimismeetodite areng on kaasa aidanud mullaorganismide tuvastamisele, mis võimaldavad määrata liike nii indiviidi kui ka koosluste tasemel. Oma doktoritöös uurisin ITS2 rakendatavust hooghännaliste (Collembola) määramisel, kuna vastava DNA lõiguga on potentsiaalselt võimalik mullaproovidest määrata samaaegselt mitmesugused eukarüootide rühmad liigi tasemele. Kuna hooghännalised on tihedalt seotud seenekooslustega (seened moodustavad olulise osa nende toidust), siis uurisin oma doktoritöös ka hooghännalistega seotud seenekoosluste ruumilist ja ajalist struktuuri kasutades nii seente kultuurides kasvatamise kui ka mass-sekveneerimise (HTS) meetodit. Vastavate HTS andmete lihtsaks ja kiireks bioinformaatiliste analüüside teostamiseks oli vaja välja töötada mass-sekveneerimisandmete töötlemise töölaud. Doktoritöö peamised tulemused ja järeldused on järgmised: 1) ITS2 lõik omab piisavat liikidevahelist erinevust, et eristada hooghännaliste liike; 2) hooghännalistega seotud seeneliikide tuvastamine on tõhusam mass-sekveneerimise meetodiga, mis tõi esile, et hooghännalised on seotud palju rohkemate seeneliikidega kui seni traditsiooniliste meetoditega kindlaks määratud; 3) tulenevalt seenekoosluste suktsessioonist on hooghännalistega seotud seenekoosluste struktuur ja liigirikkus mõjutatud nii sesoonist kui aastast; 4) töös kasutatud hooghännaliste liikide vahel ei tuvastatud toitumiseelistusi seente osas; 5) koostatud HTS andmete töötlemise programm võimaldas kiiret ja tõhusat DNA järjestuste töötlust.Microbial and faunal communities are highly diverse in soils where they play fundamental roles in several ecosystem processes ranging from soil formation to microclimate regulation. The identification of small soil organisms has benefited from the development of molecular methods that enable identification of single species to whole communities. In this thesis, I examined the usefulness of the rDNA ITS2 subregion for identification purposes of Collembola, because of its potential for simultaneous use in metabarcoding surveys of multiple taxa. Moreover, this thesis addresses the spatial and temporal structure of Collembola-associated fungal communities as based on culturing and high-throughput sequencing (HTS). To simplify the HTS data analyses, one of the objectives of this thesis was the compilation of a user-friendly and flexible platform for bioinformatics analysis of custom high-throughput amplicon sequencing data. The main results and conclusions are the following: 1) the ITS2 barcoding marker provides sufficient resolution for discriminating among Collembola species; 2) HTS outperformed the culturing method in terms of recovering Collembola-associated fungal species, and it revealed that collembolans are associated with much higher diversity of fungi than previously anticipated; 3) the Collembola-associated fungal richness and community structure exhibited significant variation in different temporal scales, which probably reflects the succession of the litter fungal community; 4) diet specialization among the studied Collembola species was not evident, suggesting that these arthropods possess relatively opportunistic feeding behavior; 5) the compiled high-throughput amplicon sequencing data analysis platform enabled efficient bioinformatics workflow for the analysis of fungal ITS2 amplicons in soil and Collembola-associated samples

The endangered northern bettong, Bettongia tropica, performs a unique and potentially irreplaceable dispersal role for truffle ectomycorrhizal fungi

Organisms that are highly connected in food webs often perform unique and vital functions within ecosystems. Understanding the unique ecological roles played by highly connected organisms and the consequences of their loss requires a comprehensive understanding of the functional redundancy among organisms. One important, yet poorly understood, food web is that between truffle‐forming ectomycorrhizal fungi and their mammalian consumers and dispersers. Mammalian fungal specialists rely on fungi as a food source, and they consume and disperse a higher diversity and abundance of fungi than do mycophagous mammals with generalist diets. Therefore, we hypothesise that mammalian fungal specialists are functionally distinct because they disperse a set of fungal taxa not fully nested within the set consumed by the combined generalist mammalian community (i.e. functional redundancy of fungal dispersal is limited). Using high‐throughput sequencing, we compared the fungal composition of 93 scats from the endangered fungal specialist northern bettong (Bettongia tropica) and 120 scats from nine co‐occurring generalist mammal species across three sites and three seasons. Compared with other generalist mammals, B. tropica consumed a more diverse fungal diet with more unique taxa. This aligns with our hypothesis that B. tropica performs a unique dispersal function for ectomycorrhizal truffle fungi. Additionally, modelling of mammalian extinctions predicted rapid loss of food web connections which could result in loss of gene flow for truffle taxa. Our results suggest that this system is sensitive to the extinction of highly connected specialist species like B. tropica and their loss could have consequences for ectomycorrhizal truffle fungal diversity. This suggests that the conservation of fungal specialists is imperative to maintaining ectomycorrhizal fungal diversity and healthy plant‐mycorrhizal relationships

Diversity and substrate-specificity of green algae and other micro-eukaryotes colonizing amphibian clutches in Germany, revealed by DNA metabarcoding

Amphibian clutches are colonized by diverse but poorly studied communities of micro-organisms. One of the most noted ones is the unicellular green alga, Oophila amblystomatis, but the occurrence and role of other micro-organisms in the capsular chamber surrounding amphibian clutches have remained largely unstudied. Here, we undertook a multi-marker DNA metabarcoding study to characterize the community of algae and other micro-eukaryotes associated with agile frog (Rana dalmatina) clutches. Samplings were performed at three small ponds in Germany, from four substrates: water, sediment, tree leaves from the bottom of the pond, and R. dalmatina clutches. Sampling substrate strongly determined the community compositions of algae and other micro-eukaryotes. Therefore, as expected, the frog clutch-associated communities formed clearly distinct clusters. Clutch-associated communities in our study were structured by a plethora of not only green algae, but also diatoms and other ochrophytes. The most abundant operational taxonomic units (OTUs) in clutch samples were taxa from Chlamydomonas, Oophila, but also from Nitzschia and other ochrophytes. Sequences of Oophila “Clade B” were found exclusively in clutches. Based on additional phylogenetic analyses of 18S rDNA and of a matrix of 18 nuclear genes derived from transcriptomes, we confirmed in our samples the existence of two distinct clades of green algae assigned to Oophila in past studies. We hypothesize that “Clade B” algae correspond to the true Oophila, whereas “Clade A” algae are a series of Chlorococcum species that, along with other green algae, ochrophytes and protists, colonize amphibian clutches opportunistically and are often cultured from clutch samples due to their robust growth performance. The clutch-associated communities were subject to filtering by sampling location, suggesting that the taxa colonizing amphibian clutches can drastically differ depending on environmental conditions

Connecting the multiple dimensions of global soil fungal diversity

How the multiple facets of soil fungal diversity vary worldwide remains virtually unknown, hindering the management of this essential species-rich group. By sequencing high-resolution DNA markers in over 4000 topsoil samples from natural and human-altered ecosystems across all continents, we illustrate the distributions and drivers of different levels of taxonomic and phylogenetic diversity of fungi and their ecological groups. We show the impact of precipitation and temperature interactions on local fungal species richness (alpha diversity) across different climates. Our findings reveal how temperature drives fungal compositional turnover (beta diversity) and phylogenetic diversity, linking them with regional species richness (gamma diversity). We integrate fungi into the principles of global biodiversity distribution and present detailed maps for biodiversity conservation and modeling of global ecological processes

How, not if, is the question mycologists should be asking about DNA-based typification

Fungal metabarcoding of substrates such as soil, wood, and water is uncovering an unprecedented number of fungal species that do not seem to produce tangible morphological structures and that defy our best attempts at cultivation, thus falling outside the scope of the International Code of Nomenclature for algae, fungi, and plants. The present study uses the new, ninth release of the species hypotheses of the UNITE database to show that species discovery through environmental sequencing vastly outpaces traditional, Sanger sequencing-based efforts in a strongly increasing trend over the last five years. Our findings chal-lenge the present stance of some in the mycological community - that the current situation is satisfactory and that no change is needed to "the code" - and suggest that we should be discussing not whether to allow DNA-based descriptions (typifications) of species and by extension higher ranks of fungi, but what the precise requirements for such DNA-based typifications should be. We submit a tentative list of such criteria for further discussion. The present authors hope for a revitalized and deepened discussion on DNA-based typification, because to us it seems harmful and counter-productive to intentionally deny the overwhelming majority of extant fungi a formal standing under the International Code of Nomenclature for algae, fungi, and plants

Structure and function of the soil microbiome underlying N2O emissions from global wetlands

Wetland soils are the greatest source of nitrous oxide (N2O), a critical greenhouse gas and ozone depleter released by microbes. Yet, microbial players and processes underlying the N2O emissions from wetland soils are poorly understood. Using in situ N2O measurements and by determining the structure and potential functional of microbial communities in 645 wetland soil samples globally, we examined the potential role of archaea, bacteria, and fungi in nitrogen (N) cycling and N2O emissions. We show that N2O emissions are higher in drained and warm wetland soils, and are correlated with functional diversity of microbes. We further provide evidence that despite their much lower abundance compared to bacteria, nitrifying archaeal abundance is a key factor explaining N2O emissions from wetland soils globally. Our data suggest that ongoing global warming and intensifying environmental change may boost archaeal nitrifiers, collectively transforming wetland soils to a greater source of N2O.The wetland soil microbiome has a major impact on greenhouse gas emissions. Here the authors characterize how a group of archaea contribute to N2O emissions and find that climate and land use changes could promote these organisms

Metabarcoding of soil environmental DNA to estimate plant diversity globally

IntroductionTraditional approaches to collecting large-scale biodiversity data pose huge logistical and technical challenges. We aimed to assess how a comparatively simple method based on sequencing environmental DNA (eDNA) characterises global variation in plant diversity and community composition compared with data derived from traditional plant inventory methods. MethodsWe sequenced a short fragment (P6 loop) of the chloroplast trnL intron from from 325 globally distributed soil samples and compared estimates of diversity and composition with those derived from traditional sources based on empirical (GBIF) or extrapolated plant distribution and diversity data. ResultsLarge-scale plant diversity and community composition patterns revealed by sequencing eDNA were broadly in accordance with those derived from traditional sources. The success of the eDNA taxonomy assignment, and the overlap of taxon lists between eDNA and GBIF, was greatest at moderate to high latitudes of the northern hemisphere. On average, around half (mean: 51.5% SD 17.6) of local GBIF records were represented in eDNA databases at the species level, depending on the geographic region. DiscussioneDNA trnL gene sequencing data accurately represent global patterns in plant diversity and composition and thus can provide a basis for large-scale vegetation studies. Important experimental considerations for plant eDNA studies include using a sampling volume and design to maximise the number of taxa detected and optimising the sequencing depth. However, increasing the coverage of reference sequence databases would yield the most significant improvements in the accuracy of taxonomic assignments made using the P6 loop of the trnL region

Regional-scale in-depth analysis of soil fungal diversity reveals strong pH and plant species effects in Northern Europe

Soil microbiome has a pivotal role in ecosystem functioning, yet little is known about its build-up from local to regional scales. In a multi-year regional-scale survey involving 1251 plots and long-read third-generation sequencing, we found that soil pH has the strongest effect on the diversity of fungi and its multiple taxonomic and functional groups. The pH effects were typically unimodal, usually both direct and indirect through tree species, soil nutrients or mold abundance. Individual tree species, particularlyPinus sylvestris,Picea abies, andPopulus x wettsteinii, and overall ectomycorrhizal plant proportion had relatively stronger effects on the diversity of biotrophic fungi than saprotrophic fungi. We found strong temporal sampling and investigator biases for the abundance of molds, but generally all spatial, temporal and microclimatic effects were weak. Richness of fungi and several functional groups was highest in woodlands and around ruins of buildings but lowest in bogs, with marked group-specific trends. In contrast to our expectations, diversity of soil fungi tended to be higher in forest island habitats potentially due to the edge effect, but fungal richness declined with island distance and in response to forest fragmentation. Virgin forests supported somewhat higher fungal diversity than old non-pristine forests, but there were no differences in richness between natural and anthropogenic habitats such as parks and coppiced gardens. Diversity of most fungal groups suffered from management of seminatural woodlands and parks and thinning of forests, but especially for forests the results depended on fungal group and time since partial harvesting. We conclude that the positive effects of tree diversity on overall fungal richness represent a combined niche effect of soil properties and intimate associations