To date, classification of anaerobic fungi has mainly relied on the use of the Internal Transcribed Spacer region 1 (ITS1) as a molecular marker of taxonomy. However, in recent years issues with the use of ITS1 as a taxonomic marker for anaerobic fungi have been revealed. This is primarily due to its size polymorphism and sequence heterogeneity, which can be substantial even within a single anaerobic fungal isolate. Previously, the anaerobic fungi network maintained and regularly updated an ITS1 database, which was based on that previously developed by Koetschan et al (2014). The last version of this database (version 3.5, released February 2020) is still available upon request, but is no longer updated. This is because it is time to move to a more robust taxonomic marker, the D1/D2 region of the large ribosomal subunit (LSU) (i.e. 28S rRNA gene).

Three New Databases

Three new databases have been developed by the anaerobic fungi network. All three databases use the same taxonomic annotation, which has been derived using the D1/D2 region of the large ribosomal subunit (LSU) (i.e. 28S rRNA gene). A published dataset containing isolates and environmental samples has been used to create the foundation of the three databases (Hanafy et al, 2020).

Please note it is not the intention of these databases to include every available anaerobic fungal sequence. These are ‘gold standard’ reference databases which contain representatives of all currently known anaerobic fungal taxa (for which there is sequence data publically available).

****Please note that the databases have not yet been updated to include all four families and the two genera that have recently been described.****

The full region sequences (i.e. ITS1 through to the D1/D2 region of the LSU) generated by Hanafy et al (2020) have been used to create a ‘full region’ database. From this database, the ITS1 and D1/D2 LSU sequence data were then extracted in order to create the basis of the respective ITS1 and LSU databases. These two draft ITS1 and LSU databases were then supplemented with additional sequences for important taxa that were not present within the full region database, generating the final ITS1 and LSU sequence databases.

The new ITS1 database is more robust than the previous one, as annotation of taxa is based on LSU. The ITS1 database is provided in order to support researchers still currently utilising ITS1 as a taxonomic marker (until they move over to D1/D2 LSU).

Details and access to the three database are given in the below table. Each database is comprised of two files: a fasta file and .txt file giving the updated taxonomy for all the sequences in the fasta file. In addition, a D1/D2 LSU alignment file is also available.

DatabaseVersionNo. of
Database files for

All sequences used in the databases come from publicly available resources, with the source origin (i.e. accession number or equivalent) indicated.

As with the previous ITS1 database, these three databases will be periodically updated as new anaerobic fungal taxa are identified.

The three databases are all compatible with Mothur. Interested in using the databases with a different pipeline such as QIIME? Please get in touch.

How to acknowledge/cite these databases?

“The AF_Full_Region/AF_ITSI/AF_LSU* database version 1.0 was obtained from the Anaerobic Fungi Network (, and was developed based on data previously published by Hanafy et al (2020).”

*Delete as appropriate depending on the database used.

Hanafy, R.A., Johnson, B., Youssef, N.H. and Elshahed, M.S., 2020. Assessing anaerobic gut fungal diversity in herbivores using D1/D2 large ribosomal subunit sequencing and multi‐year isolation. Environmental Microbiology, 22(9), pp.3883-3908.