Root-associated fungi and acquisitive root traits facilitate permafrost nitrogen uptake from long-term experimentally warmed tundra.

Root-associated fungi (RAF) and root traits regulate plant acquisition of nitrogen (N), which is limiting to growth in Arctic ecosystems. With anthropogenic warming, a new N source from thawing permafrost has the potential to change vegetation composition and increase productivity, influencing climate feedbacks. Yet, the impact of warming on tundra plant root traits, RAF, and access to permafrost N is uncertain. We investigated the relationships between RAF, species-specific root traits, and uptake of N from the permafrost boundary by tundra plants experimentally warmed for nearly three decades at Toolik Lake, Alaska. Warming increased acquisitive root traits of nonmycorrhizal and mycorrhizal plants. RAF community composition of ericoid (ERM) but not ectomycorrhizal (ECM) shrubs was impacted by warming and correlated with root traits. RAF taxa in the dark septate endophyte, ERM, and ECM guilds strongly correlated with permafrost N uptake for ECM and ERM shrubs. Overall, a greater proportion of variation in permafrost N uptake was related to root traits than RAF. Our findings suggest that warming Arctic ecosystems will result in interactions between roots, RAF, and newly thawed permafrost that may strongly impact feedbacks to the climate system through mechanisms of carbon and N cycling.

Inter-annual Persistence of Canopy Fungi Driven by Abundance Despite High Spatial Turnover.

While it is now well established that fungal community composition varies spatially at a variety of scales, temporal turnover of fungi is less well understood. Here we studied inter-annual community compositional changes of fungi in a rainforest tree canopy environment. We tracked fungal community shifts over 3 years in three substrate types (live bryophytes, dead bryophytes, and host tree bark) and compared these changes to amounts of community turnover seen at small spatial scales in the same system. The effect of substrate type on fungal community composition was stronger than that of sampling year, which was very small but significant. Although levels of temporal turnover varied among substrates, with greater turnover in live bryophytes than other substrates, the amount of turnover from year to year was comparable to what is seen at spatial distances between 5 and 9 cm for the same substrate. Stability of communities was largely driven by a few fungi with high relative abundances. A majority of fungal occurrences were at low relative abundances (≤ 0.1%). These fungi tended to be short lived and persisted to following years ≤ 50% of the time, depending on substrate. Their presence and persistence are likely impacted by stochastic processes like dispersal limitation and disturbance. Most samples contained only one or a few fungi at high relative abundance (≥ 10%) that persisted half or more of the time. These more abundant and persistent fungi are expected to have sustained functional interactions within the canopy ecosystem.

Epiphytic fungal communities vary by substrate type and at submetre spatial scales.

Fungal species have numerous important environmental functions. Where these functions occur will depend on how fungi are spatially distributed, but the spatial structures of fungal communities are largely unknown, especially in understudied hyperdiverse tropical tree canopy systems. Here we explore fungal communities in a Costa Rican tropical rainforest canopy, with a focus on local-scale spatial structure and substrate specificity of fungi. Samples of ~1 cm3 were collected from 135 points along five adjacent tree branches, with intersample distances from 1 to 800 cm, and dissected into four substrates: outer host tree bark, inner bark, dead bryophytes and living bryophytes. We sequenced the ITS2 region to characterize total fungal communities. Fungal community composition and diversity varied among substrate types, even when multiple substrates were in direct contact. Fungi were most diverse in living bryophytes, with 39% of all operational taxonomic units (OTUs) found exclusively in this substrate, and the least diverse in inner bark. Fungal communities had significant positive spatial autocorrelation and distance decay of similarity only at distances less than 1 m. Similarity among samples declined by half in less than 10 cm, and even at these short distances, similarities were low with few OTUs shared among samples. These results indicate that community turnover is high and occurs at very small spatial scales, with any two locations sharing very few fungi in common. High heterogeneity of fungal communities in space and among substrates may have implications for the distributions, population dynamics and diversity of other tree canopy organisms, including epiphytic plants.

Grass species identity shapes communities of root and leaf fungi more than elevation.

Fungal symbionts can buffer plants from environmental extremes and may affect host capacities to acclimate, adapt, or redistribute under environmental change; however, the distributions of fungal symbionts along abiotic gradients are poorly described. Fungal mutualists should be the most beneficial in abiotically stressful environments, and the structure of networks of plant-fungal interactions likely shift along gradients, even when fungal community composition does not track environmental stress. We sampled 634 unique combinations of fungal endophytes and mycorrhizal fungi, grass species identities, and sampling locations from 66 sites across six replicate altitudinal gradients in the western Colorado Rocky Mountains. The diversity and composition of leaf endophytic, root endophytic, and arbuscular mycorrhizal (AM) fungal guilds and the overall abundance of fungal functional groups (pathogens, saprotrophs, mutualists) tracked grass host identity more closely than elevation. Network structures of root endophytes become more nested and less specialized at higher elevations, but network structures of other fungal guilds did not vary with elevation. Overall, grass species identity had overriding influence on the diversity and composition of above- and belowground fungal endophytes and AM fungi, despite large environmental variation. Therefore, in our system climate change may rarely directly affect fungal symbionts. Instead, fungal symbiont distributions will most likely track the range dynamics of host grasses.

Limited overall impacts of ectomycorrhizal inoculation on recruitment of boreal trees into Arctic tundra following wildfire belie species-specific responses.

We tested whether post-fire seedling establishment of common boreal tree and expanding shrub species at treeline and in Arctic tundra is facilitated by co-migration of boreal forest mycorrhizal fungi. Wildfires are anticipated to facilitate biome shifts at the forest-tundra ecotone by improving seedbed conditions for recruiting boreal species; at the same time fire alters the composition and availability of mycorrhizal fungi critical to seedling performance. To determine the role of root-associated fungi (RAF) in post-fire seedling recruitment and future biome shifts, we outplanted four dominant boreal tree and shrub species inoculated with one of three treatments at treeline and in tundra: burned boreal forest, unburned boreal forest, or a control treatment of sterilized inoculum. We compared survivorship, growth, and physiological performance of the seedlings in relation to mycorrhizal inoculum treatment and among host species, characterized the RAF communities based on ITS-rDNA sequencing of individual root tips sampled from surviving seedlings, and tested for correlations between RAF composition and the inoculation treatments, host species, and duration of the experiment. We explored correlations between RAF composition and seedling metrics. Both live and sterile autoclaved inoculation treatments had similar effects on seedling survivorship and growth for all species. RAF composition did not vary by treatment, suggesting that most colonization was due to local fungi. However, seedling traits and growth were correlated with RAF species composition, colonization, and the relative abundance of specific RAF taxa. Picea sp. performance in particular showed strong co-variation with RAF metrics. Our results suggest that mycorrhizal co-migration is not a primary limiting factor to boreal seedling recruitment because the experimental provision of inoculum did not affect seedling recruitment; yet, RAF did influence seedling performance, particularly resident RAF at treeline and in tundra, suggesting that mycorrhizal fungi are important to vegetation processes at the treeline-tundra ecotone.

Mycobiont contribution to tundra plant acquisition of permafrost-derived nitrogen.

As Arctic soils warm, thawed permafrost releases nitrogen (N) that could stimulate plant productivity and thus offset soil carbon losses from tundra ecosystems. Although mycorrhizal fungi could facilitate plant access to permafrost-derived N, their exploration capacity beyond host plant root systems into deep, cold active layer soils adjacent to the permafrost table is unknown. We characterized root-associated fungi (RAF) that colonized ericoid (ERM) and ectomycorrhizal (ECM) shrub roots and occurred below the maximum rooting depth in permafrost thaw-front soil in tussock and shrub tundra communities. We explored the relationships between root and thaw front fungal composition and plant uptake of a 15 N tracer applied at the permafrost boundary. We show that ERM and ECM shrubs associate with RAF at the thaw front providing evidence for potential mycelial connectivity between roots and the permafrost boundary. Among shrubs and tundra communities, RAF connectivity to the thaw boundary was ubiquitous. The occurrence of particular RAF in both roots and thaw front soil was positively correlated with 15 N recovered in shrub biomass Taxon-specific RAF associations could be a mechanism for the vertical redistribution of deep, permafrost-derived nutrients, which may alleviate N limitation and stimulate productivity in warming tundra.

Altitudinal gradients fail to predict fungal symbiont responses to warming.

Climate change is shifting altitudinal species ranges, with potential to disrupt species interactions. Altitudinal gradient studies and warming experiments can both increase understanding of climate effects on species interactions, but few studies have used both together to improve predictions. We examined whether plant-fungal symbioses responded similarly to altitude and 23 yr of experimental warming. Root- and leaf-associated fungi, which can mediate plants' climate sensitivity, responded divergently to elevation vs. warming. Fungal colonization, diversity, and composition varied with altitude, but climate variables were generally weak predictors; other factors such as host plant identity, plant community composition, or edaphic variables likely drive fungal altitudinal distributions. Manipulated warming altered fungal colonization, but not composition or diversity. Leaf symbionts were more sensitive to climate and experimental warming than root symbionts. Altitudinal patterns and responses to warming differed among host plant species and fungal groups, indicating that predicting climate effects on symbioses will require tracking both host and symbiont identities. Combining experimental and observational methods can yield valuable insight into how climate change may alter plant-symbiont interactions, but our results indicate that altitude does not always serve as an adequate proxy for warming effects on fungal symbionts of plants.

Fomitopsis mounceae and F. schrenkii-two new species from North America in the F. pinicola complex.

Two new species, Fomitopsis mounceae and F. schrenkii (Polyporales, Basidiomycota) in the F. pinicola species complex in North America, are described and illustrated. Previous molecular phylogenetic analyses identified three well-delimited lineages that represent F. mounceae and F. ochracea from Canada, the Appalachian Mountains, and the northern United States and F. schrenkii from western and southwestern regions of the United States. Fomitopsis pinicola sensu stricto is restricted to Eurasia and does not occur in North America. Morphological descriptions of basidiocarps and cultures for F. mounceae, F. schrenkii, and F. ochracea are presented. The three species are readily differentiated by nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) sequence, geographic distribution, and basidiospore size. Polyporus ponderosus H. Schrenk is an earlier illegitimate synonym of F. schrenkii. Both F. mounceae and F. schrenkii have a heterothallic multiallelic incompatibility system.

The potential for mycobiont sharing between shrubs and seedlings to facilitate tree establishment after wildfire at Alaska arctic treeline.

Root-associated fungi, particularly ectomycorrhizal fungi (EMF), are critical symbionts of all boreal tree species. Although climatically driven increases in wildfire frequency and extent have been hypothesized to increase vegetation transitions from tundra to boreal forest, fire reduces mycorrhizal inoculum. Therefore, changes in mycobiont inoculum may potentially limit tree-seedling establishment beyond current treeline. We investigated whether ectomycorrhizal shrubs that resprout after fire support similar fungal taxa to those that associate with tree seedlings that establish naturally after fire. We then assessed whether mycobiont identity correlates with the biomass or nutrient status of these tree seedlings. The majority of fungal taxa observed on shrub and seedling root systems were EMF, with some dark septate endophytes and ericoid mycorrhizal taxa. Seedlings and adjacent shrubs associated with similar arrays of fungal taxa, and there were strong correlations between the structure of seedling and shrub fungal communities. These results show that resprouting postfire shrubs support fungal taxa compatible with tree seedlings that establish after wildfire. Shrub taxon, distance to the nearest shrub and fire severity influenced the similarity between seedling and shrub fungal communities. Fungal composition was correlated with both foliar C:N ratio and seedling biomass and was one of the strongest explanatory variables predicting seedling biomass. While correlative, these results suggest that mycobionts are important to nutrient acquisition and biomass accrual of naturally establishing tree seedlings at treeline and that mycobiont taxa shared by resprouting postfire vegetation may be a significant source of inoculum for tree-seedling establishment beyond current treeline.

Change in soil fungal community structure driven by a decline in ectomycorrhizal fungi following a mountain pine beetle (Dendroctonus ponderosae) outbreak.

Western North American landscapes are rapidly being transformed by forest die-off caused by mountain pine beetle (Dendroctonus ponderosae), with implications for plant and soil communities. The mechanisms that drive changes in soil community structure, particularly for the highly prevalent ectomycorrhizal fungi in pine forests, are complex and intertwined. Critical to enhancing understanding will be disentangling the relative importance of host tree mortality from changes in soil chemistry following tree death. Here, we used a recent bark beetle outbreak in lodgepole pine (Pinus contorta) forests of western Canada to test whether the effects of tree mortality altered the richness and composition of belowground fungal communities, including ectomycorrhizal and saprotrophic fungi. We also determined the effects of environmental factors (i.e. soil nutrients, moisture, and phenolics) and geographical distance, both of which can influence the richness and composition of soil fungi. The richness of both groups of soil fungi declined and the overall composition was altered by beetle-induced tree mortality. Soil nutrients, soil phenolics and geographical distance influenced the community structure of soil fungi; however, the relative importance of these factors differed between ectomycorrhizal and saprotrophic fungi. The independent effects of tree mortality, soil phenolics and geographical distance influenced the community composition of ectomycorrhizal fungi, while the community composition of saprotrophic fungi was weakly but significantly correlated with the geographical distance of plots. Taken together, our results indicate that both deterministic and stochastic processes structure soil fungal communities following landscape-scale insect outbreaks and reflect the independent roles tree mortality, soil chemistry and geographical distance play in regulating the community composition of soil fungi.

Phylogeny of Fomitopsis pinicola: a species complex.

Fungal species with a broad distribution may exhibit considerable genetic variation over their geographic ranges. Variation may develop among populations based on geographic isolation, lack of migration, and genetic drift, though this genetic variation may not always be evident when examining phenotypic characters. Fomitopsis pinicola is an abundant saprotrophic fungus found on decaying logs throughout temperate regions of the Northern Hemisphere. Phylogenetic studies have addressed the relationship of F. pinicola to other wood-rotting fungi, but pan-continental variation within F. pinicola has not been addressed using molecular data. While forms found growing on hardwood and softwood hosts exhibit variation in habit and appearance, it is unknown if these forms are genetically distinct. In this study, we generated DNA sequences of the nuc rDNA internal transcribed spacers (ITS), the TEF1 gene encoding translation elongation factor 1-α, and the RPB2 gene encoding the second largest subunit of RNA polymerase II for collections across all major geographic regions where this fungus occurs, with a primary focus on North America. We used Bayesian and maximum likelihood analyses and evaluated the gene trees within the species tree using coalescent methods to elucidate evolutionarily independent lineages. We find that F. pinicola sensu lato encompasses four well-supported, congruent clades: a European clade, southwestern US clade, and two sympatric northern North American clades. Each clade represents distinct species according to phylogenetic and population-genetic species concepts. Morphological data currently available for F. pinicola do not delimit these species, and three of the species are not specific to either hardwood or softwood trees. Originally described from Europe, F. pinicola appears to be restricted to Eurasia. Based on DNA data obtained from an isotype, one well-defined and widespread clade found only in North America represents the recently described Fomitopsis ochracea The remaining two North American clades represent previously undescribed species.

Accurate Estimation of Fungal Diversity and Abundance through Improved Lineage-Specific Primers Optimized for Illumina Amplicon Sequencing.

While high-throughput sequencing methods are revolutionizing fungal ecology, recovering accurate estimates of species richness and abundance has proven elusive. We sought to design internal transcribed spacer (ITS) primers and an Illumina protocol that would maximize coverage of the kingdom Fungi while minimizing nontarget eukaryotes. We inspected alignments of the 5.8S and large subunit (LSU) ribosomal genes and evaluated potential primers using PrimerProspector. We tested the resulting primers using tiered-abundance mock communities and five previously characterized soil samples. We recovered operational taxonomic units (OTUs) belonging to all 8 members in both mock communities, despite DNA abundances spanning 3 orders of magnitude. The expected and observed read counts were strongly correlated (r = 0.94 to 0.97). However, several taxa were consistently over- or underrepresented, likely due to variation in rRNA gene copy numbers. The Illumina data resulted in clustering of soil samples identical to that obtained with Sanger sequence clone library data using different primers. Furthermore, the two methods produced distance matrices with a Mantel correlation of 0.92. Nonfungal sequences comprised less than 0.5% of the soil data set, with most attributable to vascular plants. Our results suggest that high-throughput methods can produce fairly accurate estimates of fungal abundances in complex communities. Further improvements might be achieved through corrections for rRNA copy number and utilization of standardized mock communities. IMPORTANCE: Fungi play numerous important roles in the environment. Improvements in sequencing methods are providing revolutionary insights into fungal biodiversity, yet accurate estimates of the number of fungal species (i.e., richness) and their relative abundances in an environmental sample (e.g., soil, roots, water, etc.) remain difficult to obtain. We present improved methods for high-throughput Illumina sequencing of the species-diagnostic fungal ribosomal marker gene that improve the accuracy of richness and abundance estimates. The improvements include new PCR primers and library preparation, validation using a known mock community, and bioinformatic parameter tuning.

Nitrogen deposition alters plant-fungal relationships: linking belowground dynamics to aboveground vegetation change.

Nitrogen (N) deposition rates are increasing globally due to anthropogenic activities. Plant community responses to N are often attributed to altered competitive interactions between plants, but may also be a result of microbial responses to N, particularly root-associated fungi (RAF), which are known to affect plant fitness. In response to N, Deschampsia cespitosa, a codominant plant in the alpine tundra at Niwot Ridge (CO), increases in abundance, while Geum rossii, its principal competitor, declines. Importantly, G. rossii declines with N even in the absence of its competitor. We examined whether contrasting host responses to N are associated with altered plant-fungal symbioses, and whether the effects of N are distinct from effects of altered plant competition on RAF, using 454 pyrosequencing. Host RAF communities were distinct (only 9.4% of OTUs overlapped). N increased RAF diversity in G. rossii, but decreased it in D. cespitosa. D. cespitosa RAF communities were more responsive to N than G. rossii RAF communities, perhaps indicating a flexible microbial community aids host adaptation to nutrient enrichment. Effects of removing D. cespitosa were distinct from effects of N on G. rossii RAF, and D. cespitosa presence reversed RAF diversity response to N. The most dominant G. rossii RAF order, Helotiales, was the most affected by N, declining from 83% to 60% of sequences, perhaps indicating a loss of mutualists under N enrichment. These results highlight the potential importance of belowground microbial dynamics in plant responses to N deposition.

Towards a unified paradigm for sequence-based identification of fungi.

The nuclear ribosomal internal transcribed spacer (ITS) region is the formal fungal barcode and in most cases the marker of choice for the exploration of fungal diversity in environmental samples. Two problems are particularly acute in the pursuit of satisfactory taxonomic assignment of newly generated ITS sequences: (i) the lack of an inclusive, reliable public reference data set and (ii) the lack of means to refer to fungal species, for which no Latin name is available in a standardized stable way. Here, we report on progress in these regards through further development of the UNITE database (http://unite.ut.ee) for molecular identification of fungi. All fungal species represented by at least two ITS sequences in the international nucleotide sequence databases are now given a unique, stable name of the accession number type (e.g. Hymenoscyphus pseudoalbidus|GU586904|SH133781.05FU), and their taxonomic and ecological annotations were corrected as far as possible through a distributed, third-party annotation effort. We introduce the term 'species hypothesis' (SH) for the taxa discovered in clustering on different similarity thresholds (97-99%). An automatically or manually designated sequence is chosen to represent each such SH. These reference sequences are released (http://unite.ut.ee/repository.php) for use by the scientific community in, for example, local sequence similarity searches and in the QIIME pipeline. The system and the data will be updated automatically as the number of public fungal ITS sequences grows. We invite everybody in the position to improve the annotation or metadata associated with their particular fungal lineages of expertise to do so through the new Web-based sequence management system in UNITE.

Meeting report: fungal its workshop (october 2012).

This report summarizes a meeting held in Boulder, CO USA (19-20 October 2012) on fungal community analyses using ultra-high-throughput sequencing of the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA (rRNA) genes. The meeting was organized as a two-day workshop, with the primary goal of supporting collaboration among researchers for improving fungal ITS sequence resources and developing recommendations for standard ITS primers for the research community.

Evaluation of the authenticity of a highly novel environmental sequence from boreal forest soil using ribosomal RNA secondary structure modeling.

The number of sequences from both formally described taxa and uncultured environmental DNA deposited in the International Nucleotide Sequence Databases has increased substantially over the last two decades. Although the majority of these sequences represent authentic gene copies, there is evidence of DNA artifacts in these databases as well. These include lab artifacts, such as PCR chimeras, and biological artifacts such as pseudogenes or other paralogous sequences. Sequences that fall in basal positions in phylogenetic trees and appear distant from known sequences are particularly suspect. Phylogenetic analyses suggest that a novel sequence type (NS1) found in two boreal forest soil clone libraries belongs to the fungal kingdom but does not fall unambiguously within any known phylum. We have evaluated this sequence type using an array of secondary-structure analyses. To our knowledge, such analyses have never been used on environmental ribosomal sequences. Ribosomal secondary structure was modeled for four rRNA loci (ITS1, 5.8S, ITS2, 5' LSU). These models were analyzed for the presence of conserved domains, conserved nucleotide motifs, and compensatory base changes. Minimal free energy (MFE) foldings and GC contents of sequences representing the major fungal clades, as well as NS1, were also compared. NS1 displays secondary rRNA structures consistent with other fungi and many, but not all, conserved nucleotide motifs found across eukaryotes. However, our analyses show that many other authentic sequences from basal fungi lack more of these conserved motifs than does NS1. Together our findings suggest that NS1 represents an authentic gene copy. The methods described here can be used on any rRNA-coding sequence, not just environmental fungal sequences. As new-generation sequencing methods that yield shorter sequences become more widely implemented, methods that evaluate sequence authenticity should also be more widely implemented. For fungi, the adjacent 5.8S and ITS2 loci should be prioritized. This region is not only suited to distinguishing between closely related species, but it is also more informative in terms of expected secondary structure.

Frequent circumarctic and rare transequatorial dispersals in the lichenised agaric genus Lichenomphalia (Hygrophoraceae, Basidiomycota).

Species of the genus Lichenomphalia are mostly restricted to arctic-alpine environments with the exception of Lichenomphalia umbellifera which is also common in northern forests. Although Lichenomphalia species inhabit vast regions in several continents, no information is available on their genetic variation across geographic regions and the underlying population-phylogenetic patterns. We collected samples from arctic and subarctic regions, as well as from newly discovered subantarctic localities for the genus. Phylogenetic, nonparametric permutation methods, and coalescent analyses were used to assess phylogeny and population divergence and to estimate the extent and direction of gene flow among distinct geographic populations. All known species formed monophyletic groups, supporting their morphology-based delimitation. In addition, we found two subantarctic phylogenetic species (Lichenomphalia sp. and Lichenomphalia aff. umbellifera), of which the latter formed a well-supported sister group to L. umbellifera. We found no significant genetic differentiation among conspecific North American and Eurasian populations in Lichenomphalia. We detected high intercontinental gene flow within the northern polar region, suggesting rapid (re)colonisation of suitable habitats in response to climatic fluctuations and preventing pronounced genetic differentiation. On the other hand, our phylogenetic analyses suggest that dispersal between northern circumpolar and subantarctic areas likely happened very rarely and led to the establishment and subsequent divergence of lineages. Due to limited sampling in the Southern Hemisphere, it is currently uncertain whether the northern lineages occur in Gondwanan regions. On the other hand, our results strongly suggest that the southern lineages do not occur in the circumpolar north. Although rare transequatorial dispersal and subsequent isolation may explain the emergence of at least two subantarctic phylogenetic species lineages in Lichenomphalia, more samples from the Southern Hemisphere are needed to better understand the phylogeographic history of the genus.

Microsatellite loci development in mycoheterotrophic Corallorhiza maculata (Orchidaceae) with amplification in C. mertensiana.

PREMISE OF THE STUDY: Microsatellite primers were developed for the first time in the species Corallorhiza maculata, a nonphotosynthetic orchid that is becoming a model for studying mycorrhizal specificity. • METHODS AND RESULTS: Eight polymorphic microsatellite markers were developed using an enrichment and cloning protocol. The number of alleles for each locus ranged from two to seven. The loci were tested in three populations, and the resulting high F(ST) values suggested that these loci have utility in illuminating population structure. Cross-amplification was also achieved in the sister species C. mertensiana. • CONCLUSIONS: These microsatellite markers will be useful in further study of the population genetics of this species, including that of previously described mycorrhizal races.

A bioinformatics pipeline for sequence-based analyses of fungal biodiversity.

The internal transcribed spacer (ITS) is the locus of choice with which to characterize fungal diversity in environmental samples. However, methods to analyze ITS datasets have lagged behind the capacity to generate large amounts of sequence information. Here, we describe our bioinformatics pipeline to process large fungal ITS sequence datasets, from raw chromatograms to a spreadsheet of operational taxonomic unit (OTU) abundances across samples. Steps include assembling of reads originating from one clone, identifying primer "barcodes" or "tags," trimming vectors and primers, marking low-quality base calls and removing low-quality sequences, orienting sequences, extracting the ITS region from longer amplicons, and grouping sequences into OTUs. We expect that the principles and tools presented here are relevant to datasets arising from ever-evolving new technologies.