Intraspecific and intragenomic variability of ITS rDNA sequences reveals taxonomic problems in Ceratocystis fimbriata sensu stricto

Abstract

Fourteen new species in the Latin American Clade (LAC) of the Ceratocystis fimbriata complex recently were distinguished from C. fimbriata sensu stricto largely based on variation in ITS rDNA sequences. Among the 116 isolates representing the LAC, there were 41 ITS haplotypes. Maximum parsimony (MP) analysis of ITS sequences produced poorly resolved trees. In contrast, analyses of mating-type genes (MAT1-1-2 and MAT1-2-1) resolved a single MP tree with branches of high bootstrap and posterior probability support. Four isolates showed intragenomic variation in ITS sequences. Cloning and sequencing of PCR products from a single haploid strain identified two or more ITS sequences differing at up to 16 base positions and representing two described species. Isolates from introduced populations that appeared to be clonal based on microsatellite markers varied at up to 14 bp in ITS sequence. Strains of seven Brazilian ITS haplotypes and an isolate from Ipomoea batatas (on which the species name C. fimbriata was based) were fully interfertile in sexual crosses. These analyses support three phylogenetic species that differ in pathogenicity: C. platani, C. cacaofunesta and C. colombiana. Five ITS species (C. manginecans, C. mangicola, C. mangivora, C. acaciivora, C. eucalypticola) appear to be ITS haplotypes that have been moved from or within Brazil on nursery stock. The taxonomic status of other species delineated primarily by ITS sequences (C. diversiconidia, C. papillata, C. neglecta, C. ecuadoriana, C. fimbriatomima, C. curvata) needs further study, but they are considered doubtful species.

Molecular markers delimit cryptic species in Ceratocystis sensu stricto

“…If alternative approaches to the GCPSR are used to define species in Ceratocystis, fewer species might be resolved. This has been observed, for example, when only a single gene region, the MAT gene, and intersterility tests between individual isolates in Ceratocystis were investigated (Harrington et al 2014). There is a strong case to support the species boundaries presented in this study and accepting these will provide opportunities to better understand the biology and ecology of an important group of fungi.…”

“…Ceratocystis acaciivora and C. manginecans could not be distinguished using any of the seven markers considered in this study. These species can only be distinguished using the ITS region, which has clearly emerged as unusable since two ITS types can occur in a single isolate (Harrington et al 2014;Naidoo et al 2013), such as in C. manginecans (Al Adawi et al 2013). These data provided justification to reduce C. acaciivora to synonymy with C. manginecans in this study.…”

“…In the case of Ceratocystis, the one ITS sequence obtained from an isolate was identical to that of C. manginecans, but the second ITS type differed by seven nucleotides and had the same sequence as C. acaciivora. Whether similar problems exist for all species in Ceratocystis has not been confirmed, although it has recently been suggested for species closely related to C. acaciivora and C. manginecans (Harrington et al 2014).…”

“…Most of the cryptic species in Ceratocystis have been separated on the basis of genealogical concordance, but a few of these species have received the majority of the phylogenetic signal from the ITS region alone (Harrington et al 2014;Tarigan et al 2011;Van Wyk et al 2011a, b). The two additional gene regions used in GCPSR, βT 1 and EF 1-α, contain limited variation and provide very low or no significant bootstrap support for these species.…”

Genetic Analyses Trace the Yunnan, China Population of Ceratocystis fimbriata on Pomegranate and Taro to Populations on Eucalyptus in Brazil

“…Sequences of the ITS rDNA region (532 bp, including ITS1, 5.8S, and ITS2) and portions of the MAT2 gene (MATI-2, 1,102-bp product) and one of the MAT1 genes (MAT1-1-2, 1,022bp product) were generated as described in Harrington et al (18). Isolates were grown on MYEA (2% Difco malt extract, 0.2% Difco yeast extract, and 2% agar) for 4 to 10 days at room temperature and fighting for DNA extraction using PrepMan Ultra (Applied Biosystems, Foster City, CA).…”