This project has been in process since the spring of 2023 and is reported on iNaturalist under the username thefungiproject.

Survey Preparation

Time Calculation: A successful survey relies on appropriate triangulation of season, habitation, and weather conditions. Most fungal organisms have specific conditions that trigger their reproductive cycle. Choosing the best days to survey is mainly based on the ecosystem's moisture level over time. A good rule of thumb is that if rain has accumulated for two days at a daytime temperature above 60 degrees Fahrenheit, the possibility of finding fungi is worth the survey time. Some fungi (though few) prefer the cold and will fruit in near-freezing conditions.

Gear: Backpack, Notebook and pencil, Smartphone, TG-6 Tough Macro Digital Camera, black velvet sheet for photo backdrops, Tackle box for collection, stack of ID slips, food, and water.

Software: iNaturalist: phone application for photos and species descriptions that is used as the central project database for this project. OnX: phone application used for mapping and pathfinding. GeoForager: phone application used for rainfall calculation.

ID Slips: ID slips are printed on 3x5" index cards using a Cannon MG5320 printer using a custom application Survey Card Printer in 100 ID stacks. The ID slip issues a unique identifier for each specimen to be tracked from the field through herbarium storage. See Identifer Strategy below for more information about the ID strategy, which describes the philosophy for best use in the field and herbarium storage.

Survey Execution

Data collection: For specimens in the field, photos are collected along with morphological details, spore color, habitat description, abundance, growth pattern, and chemical reactions, among other data. Observations are also written down in a physical journal as a form of data integrity via redundancy. Two forms of observations are taken: collected and non-collected.

“Collected observation” is when the organism is given an ID slip and taken from the field to be dried and stored as a long-term herbarium specimen.

“non-collected observation” means that at least photos, geolocation, and time/date data are collected for common/already collected species to gather seasonality information, some of the most critical data points for future fruiting predictions.

For “collected observations,” the following data points were gathered:

• Photo of the mushroom is taken in situ.
• Photos are taken from different angles to gather visual morphological data.
• The specimen is then removed from the substrate, taking care to dig it out rather than “pluck it” to avoid destroying morphological features at the base.
• The specimen is photographed with a ruler and the ID card, which will be its long-term tracking number.
• Time, date, and geolocation data (latitude and longitude to at least four decimal points) are collected in iNaturalist and a physical journal to prevent data loss.
• A habitat description is generated by the mycologist
• Notable morphological data, chemical reactions, taste, smell, substrate species, associated trees for mycorrhizal species, and any other helpful identification information.

The specimen is packaged in a tackle box (or larger container) with its given ID card.

Post Survey

After the survey, specimens are unpacked from tackle boxes and bags and laid out on gridded meshes (such as a window screen) on top of precut foil squares to collect spore prints and to begin the drying process. After the spore print collection is attempted (which could take hours or days to complete), the specimens are preserved for DNA analysis and potential herbarium submission. Each specimen is desiccated in a dehydrator at 95°F until it is “cracker dry” to ensure long-term preservation. Once dried, each is sealed in a ziplock sandwich bag with its identifier card and a desiccant packet.

Further details, such as photos of spore print color, UV light reactions, chemical reactions, and microscopic features, are documented later in the home laboratory. Many fungi exhibit unique microscopic morphology, essential for distinguishing between groups and species. Sometimes, these features are the only reasonable data points for identifying species via morphology.

Hebrarium Preparation

Dried specimens in ziplock bags are stored in groups of ten in larger gallon-sized ziplock bags creating groupings AA0x, AA1x, ... AA9x for example. These larger bags are then stored in 13-quart plastic bins usually in groupings of five to ten, resulting in 50 to 100 specimens being stored in each bin. Bins are stacked a stored in a cool, dry location away from sunlight.

DNA Sequencing

To prepare material for DNA sequencing, a smaller portion of each collection will need to be packaged itself in a ziplock back with a duplicate identifier. A custom application Record Sheet Printer is loaded with a list of the specimens that will be shared to the sequencing facility and printed on 3x5" index cards using the browser's print dialog. A small portion is provided, about the size of a dime if possible of tissue from the hymenophore of the fruiting body, which generally provides the best chance of DNA.

DNA sequencing is performed at Bellevue University Science Lab. Through laboratory techniques, an ITS barcode is obtained and shared back to the project.

Bioinformatics

The DNA barcode is inputted to MycoMap and bioinformatics is run on it. From the results, we can determine if we have a match, new species, or if more work is needed. Depending on the results, the iNaturalist taxon is updated.

Nebraska Mushrooms Website

Species, higher level taxa, key, and location profiles are added to Nebraska Mushrooms regularly based on data and media from surveys, results from DNA testing.

Additional Details

Identifier Strategy

The identifier strategy is important, because a short, intelligible number is easy to write in a notebook or type in an app, asynchronous collection, print, photograph, and index in a large physical collection. The chosen identifier strategy is a four character string composed of two capital letters and two numbers:

AA##

A - Alpha

# - number

Each alpha space provides 26 spaces (A-Z) and each number space offers 10 using index-zero numbering (0-9). This provides 67,600 identifiers (AA00-ZZ99) with only four characters. This provides easy writing in notebooks with four characters, and easy groupings of 100 for herbarium storage.

Note: If the need required, an additional 16,900,00 spaces could be added by overflowing to AAA00-ZZZ00.

Advantages:

• Multiple mycologists/projects could run independently without overlapping with each other or creating confusing number gaps. For example, if two mycologists were to split up, one could take a stack of AAxx and the other could take a stack of ABxx. They would then know that those letters are designated to them without having to create a large gap in a number only system. For example, splitting up with 100-120 and 120-140.
• Random number identifiers are useful for asynchronous collection, though can be hard to store in a herbarium collection by number. The random nature doesn't account for incremental collection storage in the same way.
• Project-based collection: If the project were to require input from a community, a stack could be set aside to give identifiers to this specific project, maintaining a separate index from the main project.

Old Identifier Strategies

1-~644 - Useful for tracking in sequence, but not easy to split up in the field.

YY-rrrr - Y=Year, r=random number Easier to split up in the field, but random numbers hard to track in herbarium in sequence.