Size and sequence heterogeneity in the ITS1 of Xylaria hypoxylon isolates Gonzalo PLATAS*, Constantino RUIBAL and Javier COLLADO Centro de Investigacio ´n Ba ´sica, Merck Sharp & Dohme de Espan ˜a, S.A. Josefa Valcarcel 38, E-28027, Madrid, Spain. E-mail : gonzalo_ platas@merck.com Received 26 June 2003; accepted 14 October 2003. During a survey of 375 strains of the Xylariales, one isolate (F127076) was observed to have an ITS1 size of 833 bp. This size exceeds the average ITS1 size in the Xylariales (mean=209¡57 bp). Comparison of the DNA sequence with GenBank and with a proprietary DNA database revealed low homology with Xylaria hypoxylon ATCC 42768, and with one undescribed Xylaria species. When the ITS2 sequence was compared, these isolates were 96–98 % homologous. Sequences of other variable genes confirmed the relatedness among these strains. A closer observation of the ITS sequence of this isolate revealed the presence of three repeated domains of 250 bp plus one truncated domain, showing 52–75% homology. Sequence similarity suggests that the repeated domain is derived from the fusion of the ITS1 with a DNA fragment derived from the c-glutamyltranspeptidase gene. This example suggests that the rate of evolution of ITS1 can be independent of the rate of evolution of other genes, even when this variability is not a result of slipped strand misspairing events like in other Xylariales. This observation also indicates that recombination with other nuclear genes could participate in the evolution of the internal transcribed spacer. INTRODUCTION The Internal Transcribed Spacer (ITS) is a popular marker for phylogenetic studies because it shows a com- paratively high rate of evolution due to its non-coding structure and also because it can be easily amplified via PCR from most fungi. ITS sequences provide a sufficient level of sequence variation for phylogenetic inferences below the genus level. ITS evolution seems to be shaped by internal repetition leading to ITS size variation (Booton et al. 1999). These repetitions include repetitive elements with long repetitive units, or more commonly simple repetitive sequence motives. In these cases its evolution is subject to molecular processes such as replication slippage (Levinson & Gutman 1987), unequal crossing over (Smith 1976) and biased gene conversion (Hillis et al. 1991). In previous publications we have reported that the ITS1 rDNA of Xylariales is more prone to change than the ITS1 reported for other ascomycetes, due to the presence of a short palindrome sequence of 11 bp repeated in a tandem or quasi tandem fashion. This small sequence, due to slipped strand misspairing events, has created huge variability in the size and sequence of the ITS1 within these taxa, which affects the reliability of any phylogenetic analysis based on the ITS1 gene (Sa´ nchez-Ballesteros et al. 2000, Platas et al. 2001). To study the extent of variation of these tandem repeats, a database containing the ITS1 gene of 375 strains of Xylariales was set up. The longest ITS1 sequence from this database (830 bp) belongs to an isolate collected from the bark of a shrub from New Caledonia. Its ITS1 size is the longest found in asco- mycetes so far. This report describes this sequence, and its relatedness with two closely related isolates of Xylaria, in comparison with other variable sequences from other ribosomal and nuclear gene fragments. MATERIALS AND METHODS Fungal isolates Three isolates of Xylaria were analyzed in this study: F127076, isolated from bark of a mountainous shrub sclerophyllous forest at Prony (Goro, New Caledonia), GB6391, isolated from soil at El Bordo, Las Vigas (Veracruz, Mexico), and ATCC 42768 isolated from wood (WA, USA). The first two isolates are per- manently maintained in and available from the Merck Sharp & Dohme de Espan˜a culture collection, Madrid. * Corresponding author. Mycol. Res. 108 (1): 71–75 (January 2004). f The British Mycological Society 71 DOI: 10.1017/S0953756203008815 Printed in the United Kingdom.