Characterizing the ribosomal tandem repeat and its utility as a DNA barcode in lichen-forming fungi

Felix Grewe,Cody H. Harrison,Michael Bradshaw,Larry L. St. Clair,Steven D. Leavitt,Anne Thomas,Hanna Lindgren,H. Thorsten Lumbsch,Lucia Muggia

doi:10.1186/s12862-019-1571-4

Abstract

BackgroundRegions within the nuclear ribosomal operon are a major tool for inferring evolutionary relationships and investigating diversity in fungi. In spite of the prevalent use of ribosomal markers in fungal research, central features of nuclear ribosomal DNA (nrDNA) evolution are poorly characterized for fungi in general, including lichenized fungi. The internal transcribed spacer (ITS) region of the nrDNA has been adopted as the primary DNA barcode identification marker for fungi. However, little is known about intragenomic variation in the nrDNA in symbiotic fungi. In order to better understand evolution of nrDNA and the utility of the ITS region for barcode identification of lichen-forming fungal species, we generated nearly complete nuclear ribosomal operon sequences from nine species in the Rhizoplaca melanophthalma species complex using short reads from high-throughput sequencing.ResultsWe estimated copy numbers for the nrDNA operon, ranging from nine to 48 copies for members of this complex, and found low levels of intragenomic variation in the standard barcode region (ITS). Monophyly of currently described species in this complex was supported in phylogenetic inferences based on the ITS, 28S, intergenic spacer region, and some intronic regions, independently; however, a phylogenetic inference based on the 18S provided much lower resolution. Phylogenetic analysis of concatenated ITS and intergenic spacer sequence data generated from 496 specimens collected worldwide revealed previously unrecognized lineages in the nrDNA phylogeny.ConclusionsThe results from our study support the general assumption that the ITS region of the nrDNA is an effective barcoding marker for fungi. For the R. melanophthalma group, the limited amount of potential intragenomic variability in the ITS region did not correspond to fixed diagnostic nucleotide position characters separating taxa within this species complex. Previously unrecognized lineages inferred from ITS sequence data may represent undescribed species-level lineages or reflect uncharacterized aspects of nrDNA evolution in the R. melanophthalma species complex.

Highlights

Regions within the nuclear ribosomal operon are a major tool for inferring evolutionary relationships and investigating diversity in fungi
Assembled sequences were submitted to GenBank: accession numbers MN756803– MN756835, complete nuclear ribosomal DNA (nrDNA) cistron sequences; and MN764257-MN764288, new internal transcribed spacer (ITS) sequences generated via Sanger sequencing)
Consistent clades are recovered from alignments of different regions of nrDNA, e.g., 28S, intergenic spacer region, and intronic regions (Fig. 4) and distinct clades in the ITS are not merely idiosyncratic in the ITS region alone

Summary

Introduction

Regions within the nuclear ribosomal operon are a major tool for inferring evolutionary relationships and investigating diversity in fungi. The internal transcribed spacer (ITS) region of the nrDNA has been adopted as the primary DNA barcode identification marker for fungi. In order to better understand evolution of nrDNA and the utility of the ITS region for barcode identification of lichen-forming fungal species, we generated nearly complete nuclear ribosomal operon sequences from nine species in the Rhizoplaca melanophthalma species complex using short reads from high-throughput sequencing. Regions within the nuclear ribosomal (nrDNA) operon have been instrumental in characterizing diversity and inferring evolutionary relationships [1, 2]. The integrity of the ITS region for barcoding is questionable due to studies reporting conflicting results as to the levels of intragenomic variation found within the fungal nrDNA operon [8]. Multiple pyrosequencing-based studies suggest that distinct ITS copies are found in multiple ascomycete and basidiomycete lineages, in a relatively low proportion of sampled lineages [15, 16]

Objectives

Methods

Results

Discussion

Conclusion