Table 1 Experimental design considerations and their relevance in studies of wild animal gut mycobiota. The table describes potential resources and strategies to identify and filter non-resident gut mycobiota in amplicon sequence data

1. Metadata

Host diet

Host diet determines the likely amount of fungal contamination, e.g. omnivores should ingest more dietary fungi than carnivores

Niche specialisation

Dietary specialisation determines the potential diversity of fungal contamination, e.g. generalists may ingest a higher diversity of environmental fungi than specialists

Developmental stage

Host diet may change during development, e.g. adult mammals have a higher likelihood of ingesting fungi than neonates (that consume milk)

Season

Both host diet and the type and availability of fungi can change with season

2. Sampling scheme

Longitudinal sampling

A time series analysis of the gut mycobiota can be used to define a set of core fungal sequence variant (SV) groups (especially in animals whose dietary fungi are seasonally available)

Spatial sampling

Increasing the spatial extent of sampling can help to identify the core fungal SVs based on frequency of occurrence

Time that sample is exposed to the environment

Fungi may rapidly colonise and grow on faeces after defecation (a more prominent risk for non-invasive sampling)

Time since sample collection

Time between sample collection and preservation determines the potential for overgrowth of certain fungi

Source tracking

Information about fungi that inhabit the host's environment and/or diet can be used to identify the taxa that have the potential to be commonly ingested

Negative/positive controls

A diverse community of microbes can be present within the laboratory and the reagents, and contamination of samples by these microbes should be avoided as much as possible to maximise the signal to noise ratio in the data

Sterile work

3. Bioinformatic analyses

Positive filter using reference database(s), e.g. Targeted Host Fungi database

Fungal SVs that reside in the digestive systems of other animals are likely also to inhabit wild animal's gut. Note that reference databases are not always correct or up to date. The author(s) must clearly state the version of a database used in their analyses

Filter SVs using fungal traits database(s), e.g. FUNGuild

Fungal SVs that have certain traits or growth forms, e.g. being 'macrofungi', are unlikely to be authentic residents of the gut microbiota

Comparative analysis

Captive and/or laboratory animals often have less opportunities to ingest a diverse community of fungi, and these data may allow the likely ingested fungal SVs to be identified in wild conspecifics