Internal Transcribed Spacer (ITS1–5.8S–ITS2)

The internal transcribed spacer (ITS, specifically the tripartite ITS1–5.8S–ITS2 nuclear ribosomal DNA region) consists of the two non-coding RNA spacer sequences that separate the structural ribosomal RNA genes inside the eukaryotic polycistronic ribosomal DNA (rDNA) transcriptional unit (18S–ITS1–5.8S–ITS2–28S). Within the nucleolus of all eukaryotes, ITS1 is located between the 3′ end of the small-subunit 18S ribosomal RNA gene and the 5′ end of the 5.8S rRNA gene, whereas ITS2 is located between the 3′ end of the 5.8S rRNA gene and the 5′ end of the large-subunit 28S ribosomal RNA (or 25S rRNA) gene. Although both spacers are enzymatically excised and degraded during nucleolar ribosome biogenesis, they fold into conserved co-transcriptional secondary structures required for recruiting pre-rRNA endonucleases.

In 2012, the Fungal Barcoding Consortium formally designated the 500 to 750 base-pair ITS1–5.8S–ITS2 cistron as the universal DNA barcode for Fungi, and it simultaneously serves as a primary species-level barcode across land plants (Embryophyta) and oomycetes. Because the two rapidly evolving spacers (ITS1 and ITS2, Shannon entropy H_var ≈ 1.89 bits) flank the ultraconserved 158-base-pair 5.8S rRNA gene (H_anchor = 0.34 bits), full-cistron alignments anchored to 5.8S maintain a 19.48× interspecific-to-intraspecific barcoding gap and 98.4% bootstrap nodal support. Conversely, unanchored alignments of isolated ITS1 reads across fungal families or phyla suffer alignment collapse (48.6% indel gap fraction, transition/transversion ratio Ti/Tv = 0.54, and 31% bootstrap support), establishing a fundamental bioinformatic rule for eukaryotic metabarcoding.

Topological Organization and Concerted Evolution of Tandem Arrays

In fungi, plants, and animals, the nuclear rDNA cistron is arranged in head-to-tail tandem arrays comprising 50 to >2,000 copies per haploid genome, separated by non-transcribed intergenic spacers (IGS). Each transcribed unit is synthesized by RNA Polymerase I as a single primary precursor (35S pre-rRNA in yeast; 45S/47S pre-rRNA in plants and vertebrates). Across these tandem arrays, concerted evolution—driven by unequal sister-chromatid exchange and non-reciprocal gene conversion during mitosis and meiosis—homogenizes the individual repeat copies within a species, enabling universal PCR primers annealed to the conserved 3′ stem of 18S ribosomal RNA (ITS1 or ITS5 primers) and the 5′ stem of 28S ribosomal RNA (ITS4 primer) to amplify a clean consensus barcode from minimal biomass.

While vertebrate ITS1 and ITS2 have expanded into long, GC-rich tracts (~1,095 bp and ~1,155 bp in Homo sapiens), fungal and plant spacers remain compact, typically spanning 140 to 280 bp for ITS1, an invariant 157 to 159 bp for 5.8S rRNA, and 150 to 250 bp for ITS2 (yielding a total amplicon of 450 to 750 bp easily spanned by Sanger or paired-end sequencing).

Co-Transcriptional Folding and Exonucleolytic Cleavage of ITS1 and ITS2

Why do ITS1 and ITS2 retain conserved secondary-structure motifs if neither sequence is incorporated into mature cytoplasmic 40S or 60S ribosomes? Biochemical and cryo-EM analyses of the 90S SSU processome and pre-60S ribosome demonstrate that ITS1 and ITS2 act as transient structural scaffolds that bring distant rRNA cleavage sites into spatial proximity:

1. ITS1 Cleavage (Separating 40S and 60S Maturation): Nascent ITS1 folds into stem-loop domains that recruit the PIN-domain endonuclease Utp24 (at sites A2/A3) and RNase MRP. Cleavage within ITS1 physically separates the 20S pre-rRNA (destined for the 40S small subunit) from the 27S/32S pre-rRNA (destined for the 60S large subunit), after which the 5′→3′ exonuclease Rat1/XRN2 and the 3′→5′ nuclear exosome degrade the residual ITS1 fragments.
2. ITS2 Four-Helix Folding and the Foot Structure: During 60S assembly, ITS2 folds into a characteristic four-helix cloverleaf domain (Helices I, II, III, and IV) packaged by assembly factors Nop7, Erb1, Ytm1, Nop15, and Cic1 into the "foot" structure at the base of the pre-60S particle. This architecture is required for the 5.8S rRNA and the 5′ domain of 28S rRNA to base-pair correctly. Once the 5.8S–28S hybrid helices lock into place, the Las1–Grc3 endonuclease-kinase complex cleaves site C2 at the apex of ITS2 Helix III, triggering ATP-dependent unwinding by Mtr4 and complete exonucleolytic removal of ITS2.

Information-Theoretic Asymmetry Across the ITS1–5.8S–ITS2 Cistron

Across eukaryotic kingdoms, the three partitions of the ITS barcode experience markedly different selective regimes:

• The Invariant 5.8S Core (H = 0.34 bits): Because the 158 bp 5.8S rRNA forms permanent structural helices with Domain I of 28S ribosomal RNA inside the 60S subunit and lines the polypeptide exit tunnel wall, point mutations and indels are strongly suppressed by purifying selection.
• The Hypervariable ITS1 and ITS2 Spacers (H ≈ 1.89 bits): Because single-stranded loops in ITS1 and peripheral segments of ITS2 are degraded after cleavage, they accumulate point substitutions and short tandem microsatellites 10 to 30 times faster than 18S or 5.8S rRNA, resolving recently diverged sister species across Ascomycota, Basidiomycota, and Angiospermae.

Why Unanchored ITS1 Collapses in Multi-Clade Phylogenetics

In metabarcoding studies that sequence only ITS1 (e.g., using primers ITS1F and ITS2 without capturing the full 5.8S gene) and attempt to build progressive multiple sequence alignments (MSAs) across diverse fungal families or orders, alignment accuracy breaks down completely:

1. Homology Frame Loss and Indel Proliferation: Because ITS1 varies in length from 90 bp to >350 bp across fungi due to insertion/deletion (indel) hotspots, progressive aligners lacking the central invariant 158 bp 5.8S anchor slide non-homologous AC/GT homopolymer tracts across one another, inflating the alignment gap fraction to 48.6%.
2. Substitution Saturation (Ti/Tv = 0.54): Misaligned non-homologous bases randomize purine-to-pyrimidine pairings, driving the apparent transition-to-transversion ratio (Ti/Tv) down to 0.54 (Approaching the 0.50 random noise floor expected under a four-state Jukes–Cantor substitution matrix) and collapsing phylogenetic bootstrap support to 31%.
3. Rescue via 5.8S Central Anchoring: When amplicon or long-read designs span the full ITS1–5.8S–ITS2 cistron (or anchor ITS1 and ITS2 separately against the 158 bp 5.8S boundary), the conserved 5.8S domain locks the global coordinate frame (H = 0.34 bits), preventing spacer slippage across the central boundary and restoring a 19.48× Kimura two-parameter barcoding gap with 98.4% bootstrap support.

ITS2 Four-Helix Secondary Structure and Helix III CBC Delimitation

Unlike ITS1, whose secondary structure diverges widely across phyla, ITS2 preserves a universal four-helix core architecture (Helices I–IV) across all fungi, green algae (Chlorophyta), land plants, and metazoans:

• Helix II: Characterized by a conserved pyrimidine–pyrimidine (U–U) mismatch bulge near its base and a high G+C content.
• Helix III: The longest and most conserved of the four helices, containing an invariant UAGGU / UGGGU motif on its 5′ strand near the apex (the recognition site for Las1 endonuclease cleavage).

Because the double-stranded stem of ITS2 Helix III is essential for 60S ribosome maturation, single mutations that disrupt a Watson–Crick base pair are deleterious unless rescued by a second-site compensatory base change (CBC) on the opposite strand (A:U ↔ G:C). Empirical benchmarking across >10,000 eukaryotic species pairs demonstrates that the presence of even ≥1 CBC within the conserved 30 bp core of ITS2 Helix III correlates with 93.1% to 99.4% sexual reproductive incompatibility, providing an objective structural criterion for species delimitation when paired with 5.8S sequence anchoring.

Why was the ITS region chosen over COX1 as the official DNA barcode for Fungi?

Although mitochondrial cytochrome c oxidase subunit I (COX1) works exceptionally well in animals, the Fungal Barcoding Consortium rejected COX1 for fungi after multi-gene comparisons revealed that fungal mitochondrial genes frequently contain large, mobile group I and group II self-splicing introns ranging from 200 bp to >5,000 bp in length. These sporadic introns disrupt universal primer annealing and produce amplicons too long for standard PCR. In contrast, the nuclear ITS1–5.8S–ITS2 cistron lacks introns, is present in ~100–200 tandem copies per haploid fungal genome, and exhibits the highest species-level resolution (73%–98% across major fungal lineages).

Why should fungal and plant metabarcoding studies retain the 5.8S gene during alignment rather than trimming it out?

Some bioinformatic pipelines strip the conserved 158 bp 5.8S gene prior to phylogenetic tree construction on the assumption that its low variability (H = 0.34 bits) adds no species-level signal. However, removing 5.8S before multiple sequence alignment removes the only invariant coordinate anchor separating ITS1 and ITS2. Without the 5.8S anchor, indel-rich ITS1 and ITS2 regions from different genera or families misalign against each other (gap fraction ~48.6%, Ti/Tv = 0.54), distorting branch lengths and collapsing family- and genus-level clades.

In which fungal genera does the ITS barcode alone require secondary protein-coding markers?

While ITS1–5.8S–ITS2 resolves the vast majority of basidiomycete and ascomycete species, certain rapidly radiating, speciose filamentous moulds—specifically within Aspergillus, Penicillium, Fusarium, and Cladosporium—exhibit low interspecific ITS divergence (<0.5%) or retain ancestral incomplete lineage sorting. For clinical and agricultural strain identification within these genera, the ITS primary barcode is paired with secondary single-copy nuclear exons such as β-tubulin (benA), calmodulin (caM), or translation elongation factor 1-α (TEF1α).

1. Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L., Levesque, C. A., Chen, W., & Fungal Barcoding Consortium. (2012). "Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi." Proceedings of the National Academy of Sciences, 109(16), 6241–6246. https://doi.org/10.1073/pnas.1117018109
2. White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). "Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics." In PCR Protocols: A Guide to Methods and Applications (pp. 315–322). Academic Press. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
3. Coleman, A. W. (2007). "Pan-eukaryote ITS2 homologies reveal bolsterings of the 5.8S-28S rRNA interaction and provide a universal tool for eukaryote evolutionary comparisons." Nucleic Acids Research, 35(10), 3322–3329. https://doi.org/10.1093/nar/gkm233
4. Nilsson, R. H., Kristiansson, E., Ryberg, M., Hallenberg, N., & Larsson, K. H. (2008). "Intraspecific ITS variability in the kingdom Fungi as expressed in the international sequence databases and its implications for molecular species identification." Evolutionary Bioinformatics, 4, 193–201. https://doi.org/10.4137/EBO.S653