Fungi in Thailand: A Case Study of the Efficacy of an ITS Barcode for Automatically Identifying Species within the Annulohypoxylon and Hypoxylon Genera

Abstract

Thailand, a part of the Indo-Burma biodiversity hotspot, has many endemic animals and plants. Some of its fungal species are difficult to recognize and separate, complicating assessments of biodiversity. We assessed species diversity within the fungal genera Annulohypoxylon and Hypoxylon, which produce biologically active and potentially therapeutic compounds, by applying classical taxonomic methods to 552 teleomorphs collected from across Thailand. Using probability of correct identification (PCI), we also assessed the efficacy of automated species identification with a fungal barcode marker, ITS, in the model system of Annulohypoxylon and Hypoxylon. The 552 teleomorphs yielded 137 ITS sequences; in addition, we examined 128 GenBank ITS sequences, to assess biases in evaluating a DNA barcode with GenBank data. The use of multiple sequence alignment in a barcode database like BOLD raises some concerns about non-protein barcode markers like ITS, so we also compared species identification using different alignment methods. Our results suggest the following. (1) Multiple sequence alignment of ITS sequences is competitive with pairwise alignment when identifying species, so BOLD should be able to preserve its present bioinformatics workflow for species identification for ITS, and possibly therefore with at least some other non-protein barcode markers. (2) Automated species identification is insensitive to a specific choice of evolutionary distance, contributing to resolution of a current debate in DNA barcoding. (3) Statistical methods are available to address, at least partially, the possibility of expert misidentification of species. Phylogenetic trees discovered a cryptic species and strongly supported monophyletic clades for many Annulohypoxylon and Hypoxylon species, suggesting that ITS can contribute usefully to a barcode for these fungi. The PCIs here, derived solely from ITS, suggest that a fungal barcode will require secondary markers in Annulohypoxylon and Hypoxylon, however. The URL http://tinyurl.com/spouge-barcode contains computer programs and other supplementary material relevant to this article.

Identification of a Fungal 1,8-Cineole Synthase from Hypoxylon sp. with Specificity Determinants in Common with the Plant Synthases

“…This clade is clearly resolved from other genera, Nemania, Xylaria, and Biscogniauxia, by both maximum likelihood and Bayesian phylogenetic approaches. The genera containing cineole producers are closely related, and although their phylogenetic relationship was not resolved by ITS sequencing alone in our tree, consistent with previous publications, we can observe to which of these groups our isolates are most closely related (49).…”

“…The primers used for amplification were ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCT-TATTGATATGC), and the resulting PCR products were sequenced by the W. M. Keck Foundation (48). A phylogenetic tree was constructed comparing the ITS sequence of cineoleproducing isolates from this study with previous cineole producers (CI-4, Ni25-2A, and EC12) and taxa reported in several recent molecular phylogenetic studies of genera in the family Xylariaceae (Table 1) (49,50). Sequences were aligned using MUSCLE version 3.8.31, and the phylogenetic tree was constructed using Bayesian (MrBayes version 3.2.0) and maximum likelihood methods (RAxML version 7.2.8) (51,52).…”

Figures of merit and statistics for detecting faulty species identification with DNA barcodes: A case study in Ramaria and related fungal genera

“…Phylogenetic trees were rooted with two sequences under Gloeocantharellus persicinus and Gloeocantharellus purpurascens. Gaps were treated as missing data [13]. Branch lengths equal to zero were collapsed to polytomies.…”

Species delimitation in Cladonia (Ascomycota): a challenge to the DNA barcoding philosophy

“…Probability of Correct Identification (PCI) was calculated according Suwannasai et al . (), using the previous aligments. The p‐Distance (also known as ‘uncorrected distance’, the fraction of aligned nucleotide pairs that are not identical pairs) was used to calculate the PCI values.…”