Systematic Entomology (2007), 32, 420–428 DOI: 10.1111/j.1365-3113.2007.00385.x Double trouble for grasshopper molecular systematics: intra-individual heterogeneity of both mitochondrial 12S-valine-16S and nuclear internal transcribed spacer ribosomal DNA sequences in Hesperotettix viridis (Orthoptera: Acrididae) GREGORY A. SWORD 1,2 ,LAURA B. SENIOR 1 ,JOHN F. GASKIN 1 and A N T H O N Y J O E R N 3 1 Northern Plains Agricultural Research Laboratory, United States Department of Agriculture, Agricultural Research Service, Sidney, Montana, U.S.A., 2 School of Biological Sciences, The University of Sydney, NSW, Australia. 3 Division of Biology, Kansas State University, Manhattan, Kansas, U.S.A. Abstract. Hesperotettix viridis grasshoppers (Orthoptera: Acrididae: Melanoplinae) exhibit intra-individual variation in both mitochondrial 12S-valine-16S and nuclear internal transcribed spacer (ITS) ribosomal DNA sequences. These findings violate core assumptions underlying DNA sequence data obtained via polymerase chain reaction (PCR) amplification for use in molecular systematics investigations. Undetected intra-individual variation of this sort can confound phylogenetic analyses at a range of taxonomic levels. The use of a DNA extraction protocol designed to enrich mitochondrial DNA as well as an initial long PCR of approximately 40% of the grasshopper mitochondrial genome failed to control for the presence of paralogous mitochondrial DNA-like sequences within individuals. These findings constitute the first demonstration of intra-individual heterogeneity in mitochondrial DNA-like sequences in the grasshopper subfamily, Melanoplinae, and only the second report of intra-individual variation in nuclear ITS ribosomal DNA sequences in grasshoppers. The fact that intra-individual variation was detected in two independent DNA marker sets in the same organism strengthens the notion that the orthology of PCR-derived DNA sequences should be examined thoroughly prior to their use in molecular phylogenetic analyses or as DNA barcodes. Introduction The rapid evolution and ease of polymerase chain reaction (PCR) amplification of both mitochondrial genes (mtDNA) and internal transcribed spacer regions (ITS1 and 2) of nuclear ribosomal DNA (rDNA) have facilitated the use of these sequences as genetic markers in numerous population- level phylogeographical and species boundary investigations (Hillis & Dixon, 1991; Simon et al., 1994; Hillis et al., 1996; Avise, 2004). Two important assumptions underlying the use of PCR amplification and direct DNA sequencing in phylogenetic analyses are that: (1) the PCR product obtained is the desired target sequence, and (2) all of the amplified DNA sequences represent a single orthologous locus that is invariant within individuals, but homologous due to common ancestry with sequences obtained from other individuals (Palumbi, 1996; Sanderson & Shaffer, 2002). A growing number of examples indicate that these assumptions are routinely violated due to the presence of intra-individual variation in DNA sequences that share homology as a result of gene duplication events rather than common ancestry. If undetected, the amplification and sequencing of these paralogous loci can potentially con- found molecular phylogenetic analyses (Sorenson & Quinn, 1998; Bensasson et al., 2001a; A ´ lvarez & Wendel, 2003; Funk & Omland, 2003). Correspondence: G. A. Sword, School of Biological Sciences, The University of Sydney, Macleay Building A12, Sydney, NSW 2006, Australia. E-mail: greg.sword@bio.usyd.edu.au 420 # 2007 The Authors Journal compilation # 2007 The Royal Entomological Society