Assessing the effect of varying sequence length on DNA barcoding of fungi

Abstract

DNA barcoding shows enormous promise for the rapid identification of organisms at the species level. There has been much recent debate, however, about the need for longer barcode sequences, especially when these sequences are used to construct molecular phylogenies. Here, we have analysed a set of fungal mitochondrial sequences - of various lengths - and we have monitored the effect of reducing sequence length on the utility of the data for both species identification and phylogenetic reconstruction. Our results demonstrate that reducing sequence length has a profound effect on the accuracy of resulting phylogenetic trees, but surprisingly short sequences still yield accurate species identifications. We conclude that the standard short barcode sequences ( approximately 600 bp) are not suitable for inferring accurate phylogenetic relationships, but they are sufficient for species identification among the fungi.

Fig. 1

NJ trees based on relatively long mitochondrial sequences. Panel a, this tree was constructed with the concatenated cDNA sequences from five mitochondrial genes (see Methods). Panel b, this shows an NJ tree based on the predicted amino acid sequences of the same five genes. Areas where the two trees differ are highlighted in red. Clades are labelled as follows: A1, Ascomycota Saccharomyces; A2, Ascomycota Schizosaccharomyces; A3, Ascomycota Sordariomycetes; A4, Ascomycota Eurotiomycetes; B, Basidiomycota; C, Chytridiomycota.

Fig. 2

NJ trees based on the sequence of a single mitochondrial gene, cox1. Panel a shows the DNA-based tree and Panel b shows the protein tree. Differences in topology between the two trees are highlighted in red. Clades are labelled as in Fig. 1.

Fig. 3

NJ trees based on the barcode region of the cox1 gene. Panel a shows the tree based on the barcode DNA sequence (∼600 bp), while Panel b shows the tree based on the predicted amino acid sequences. Differences in topology between the two trees are highlighted in red. Clades are labelled as in Fig. 1. • indicates the amino acid sequences are identical in the two species on that branch.

Fig. 4

Nucleotide variation within the barcode region in seven strains Aspergillus from three closely related species. The GenBank Accession nos of the sequences used for generating the barcodes are AF178445 (As.nige.1), NC_007445 (As.nige.2), AF178444 (As.nige.3), AF178443 (As.nige.4), NC_007597 (As.tubi.1), AY802759 (As.tubi.2) and X00790(As.nidu). There are a total of 36 variant sites, consisting of 8 nonsynonymous sites (upper case in the consensus sequence) and 28 synonymous sites.

Fig. 5

Extended barcode sequences have increased resolving power. Panel a shows an NJ tree constructed with the standard barcode sequence (∼600 bp). Panel b shows a tree based on an extended barcode sequence (∼1200 bp). • indicates the DNA sequences are identical in the two strains on that branch. Sequence accession nos are, AF178445 (n1), NC_007445 (n2), AF178444 (n3), AF178443 (n4), NC_007597 (t1), AY802759 (t2), X00790(As.nidu.), NC_004336 (grubii1), AY560510 (grubii2) and AY560610 (neoformans).