What a DNA barcode is supposed to do
A DNA barcode is a short, standardized stretch of DNA that can be read from many individuals and compared against a reference collection. A good barcode varies little within a species but differs clearly between species, so that an unknown sequence can be matched to a name or at least to a defined group. Barcodes help most with organisms that are hard to identify by eye.
AM fungi live inside plant roots and in soil, form spores that are often small and share few distinguishing features, and cannot be grown without a host plant. Many species are therefore recognized, counted and compared only through their DNA. Choosing the right marker is consequently a central question for the whole field.
The ribosomal RNA gene region
Most barcodes for fungi come from the ribosomal RNA gene cluster, which is present in many copies in the genome and is easy to amplify. The cluster contains, in order, the small subunit (SSU) gene, a first internal transcribed spacer (ITS1), the 5.8S gene, a second spacer (ITS2) and the large subunit (LSU) gene. The coding genes evolve slowly and are conserved across wide groups, while the spacers evolve faster and carry more differences between close relatives.
For many fungal groups the ITS region has become the standard barcode. In AM fungi the situation is more complicated, because the ribosomal region can vary even within a single spore, a point that is discussed below. This has led researchers to look at other parts of the cluster, and one of them is the D2 region of the LSU gene.
The LSU D2 region
Where it sits
The LSU gene contains stretches that are highly conserved and stretches that vary more, called divergent domains. The D2 domain is one of these variable stretches. It is short enough to be read in a single sequencing reaction, yet variable enough to differentiate many species.
Why it works for AM fungi
For AM fungi, the D2 region offers a useful balance. It is easier to align across distantly related lineages than the spacers, which can be so variable that sequences from different genera cannot be lined up reliably. At the same time it still shows differences between many closely related species. Its focus is the reason why the species list on this site uses the LSU D2 region as the barcode behind its identifiers.
What it cannot do
No single short marker is perfect. In some groups, closely related species share identical or nearly identical D2 sequences, and in others a single species may show variation. A barcode result is therefore strong evidence, not proof, and is best interpreted together with other information.
The SSU-ITS-LSU fragment
A short marker has one serious disadvantage: it holds limited phylogenetic information. For this reason the group behind this site established a longer fragment as a barcoding marker for AM fungi more than a decade ago. The fragment spans the end of the SSU gene, the complete ITS region including the 5.8S gene, and the beginning of the LSU gene, including the D2 domain.
The advantages of the longer fragment are practical.
- It combines conserved and variable parts, so that it supports both broad placement in the tree and fine-scale comparison between close relatives.
- It can be amplified and sequenced as a unit, which avoids the problem of joining separate short sequences that may not come from the same organism.
- It links environmental sequences to named fungi, because the same fragment can be obtained from a described culture and from a root sample.
Why the combination matters for environmental studies
Many AM fungi are known only from environmental samples, never from spores. Sequences from such studies are sometimes short, come from different parts of the ribosomal region in different projects, and cannot easily be compared. When a project uses the SSU-ITS-LSU fragment, its sequences can be placed directly against those of described species and against those from other environmental surveys.
The identifiers on this site, known as EP numbers, are given both to species characterized by the LSU D2 barcode and to species known from environmental studies only, based on the longer fragment. In this way an uncultured lineage can be named in a stable way, and if a culture or a spore is found later, it can be connected to the existing identifier.
Limits and practical choices
Intragenomic variation
The ribosomal region is present as many copies within a genome, and in AM fungi these copies are not always identical. Consequently, a single spore can contain more than one sequence variant. This does not make the markers useless, but it does mean that any barcode study should sample variation honestly. A description that reports a single sequence per species gives no way to judge how far its variants extend.
A sound approach is to clone and sequence several copies, to examine how they cluster, and to report the range of variation.
Reference data and naming
A barcode becomes useful only when its sequences are anchored to organisms that have been properly identified. For AM fungi this means sequences obtained from well-characterized cultures or from type material, linked to a formal description. Without that anchor, a sequence is merely a string of letters, and a database match to another unnamed sequence tells the user nothing new.
This is also where problems arise. If a reference sequence was deposited under a wrong name, every later match inherits the error. For that reason the species list on this site connects names to sequence-based identifiers and to the formal descriptions, so that users can check the chain from a DNA sequence back to a described fungus.
Choosing between a short and a long marker
The decision between the D2 region alone and the longer fragment depends on the question. For a quick check of whether a culture matches a known species, a short marker can be enough, especially when the reference library is dense. For describing a new species, placing an unknown lineage, or judging relationships among genera, the longer fragment is the better tool because it supplies more characters for analysis.
Practical advice for using the barcode
- Use a marker that is well represented in the reference data you plan to compare with, since a barcode is only as useful as its reference library.
- Prefer sequences long enough to be aligned and placed in a phylogenetic tree, not only matched by a similarity score.
- Check how a published species was defined: how many specimens, how many sequences, and whether the variation within the species was described.
- Treat similarity thresholds with caution, because the amount of variation between species differs from one lineage to the next.
- Combine the barcode result with morphology and with other genes wherever you can.
Summary
In short, choose the marker to fit the question.
The LSU D2 region is a compact, informative barcode that suits the identification of many AM fungi, and the longer SSU-ITS-LSU fragment extends this approach to uncultured lineages and gives more phylogenetic depth. Neither marker removes the need for careful sampling and critical interpretation. Used with those habits, they provide a firm base for naming, tracking and comparing the fungi in the roots of most land plants.
