Intragenomic Variation Within ITS1 and ITS2 of Freshwater Crayfishes (Decapoda: Cambaridae): Implications for Phylogenetic and Microsatellite Studies

D. James Harris 0 1 2 Keith A. Crandall 0 2 0 partment of Zoology and Monte L. Bean Museum, Brigham Young University , 574 Widtsoe Building, Provo, Utah 84602-5255 1 Present address: Unidade de Gene tica Animal e Conserva c oa , Campus Agra rio de Vaira o, R. Monte-Crasto , Portugal 2 Department of Zoology and Monte L. Bean Museum, Brigham Young University Intragenomic variation in ITS1 and ITS2 is known to exist but is widely ignored in phylogenetic studies using these gene regions. The amount of variation in seven crayfish species, including three populations of Orconectes luteus and two of Procambarus clarkii, was assessed by sequencing 3, 5, or 10 clones from the same individuals, for a total of 77 sequences. The ITS1 and ITS2 sequences reported here are some of the longest known, with aligned lengths of 760 and 1,300 bp, respectively. They contain multiple microsatellite insertions, all of which show considerable intragenomic variation in the number of repeat elements. This variation is enough to obscure phylogenetic relationships at the population level, although relationships between species can be estimated. Given the hybridization techniques used to locate microsatellites, multiple-copy regions like ITS1 and ITS2 will be preferentially found if they contain microsatellites, and in these cases the microsatellites will not behave as typical Mendelian markers and could give spurious results. Introduction The eukaryotic ribosomal DNA (rDNA) array typically consists of several hundred tandemly repeated copies of the transcription unit, which encodes 18S, 5.8S, and 28S genes, with two internal transcribed spacers, ITS1 and ITS2 (fig. 1). Nuclear rDNA sequences have been widely used in estimating phylogenies for many organisms, with ITS1 being particularly widely used at the population and species level due to its high level of sequence variation (Vogler and DeSalle 1994; Miller, Crabtree, and Savage 1996; Fabry, Ko hler, and Coleman 1999; Schulenberg, Englisch, and Wagele 1999). Intragenomic rDNA diversity is generally low due to concerted evolution (Brown, Wensink, and Jordan 1972)individual repeats in the multigene family evolve in concert, resulting in the homogenization of all the repeats in an array. Although this appears to be the norm, variation within an individual is known (e.g., Carranza et al. 1996; Hugall, Stanton, and Mortiz 1999). Whenever concerted evolution is slower than speciation, a single genome will contain divergent paralogs. Vogler and DeSalle (1994) showed that sequence variation in ITS1 within individual tiger beetles, Cicindela dorsalis, was high, although they still exhibited phylogenetic separation coinciding with geographic separation of populations. Wesson et al. (1992) reported 0.46% variation within 10 clones of ITS2 from a single mosquito, Aedes simpsoni, while intraspecific variation in Aedes aegypti was only 1.17%. We investigated levels of intragenomic variation within the ITS1 and ITS2 regions of freshwater crayfish (Decapoda; Cambaridae). All of the individuals studied, both from different species and from different genera, showed some level of intragenomic variation in sequence composition of ITS1 and ITS2. Although separation between species was well supported, variation within individuals was greater than any differentiation among populations, making these sequences uninformative at this level. Intraspecific variation was primarily due to the presence of a number of microsatellite loci within these regions, which show considerable variation in the number of repeats within individuals. The presence of microsatellites is well documented in many multigene families, such as the human rRNA genes (Gonzalez et al. 1990) and the primate RNU2 locus (Liao and Weiner 1995). However, in the absence of breeding studies, microsatellite loci are typically statistically summarized as codominant Mendelian markers, something they are clearly not if they are found in these regions. Implications are therefore important to both microsatellite studies and phylogenetic analyses using ITS sequences. Materials and Methods Crayfish Samples The following crayfish specimens were examined: Orconectes luteus (four individuals, three separate populations), Orconectes macrus, Orconectes neglectus, Orconectes punctimanus, Orconectes longidigitus, Orconectes virilis, and Procambarus clarkii (2 individuals). All of the specimens were collected by hand or net (table 1). We used these specimens because they have a well-defined phylogeny based on 16S rDNA sequences (Crandall and Fitzpatrick 1996), and the populations of O. luteus are easily discernable using 16S rDNA and AFLP data (Fetzner and Crandall 1999). Upon capture, crayfish were identified, and a tissue sample was taken and preserved in liquid nitrogen until it was placed in permanent storage at 2808C. The remainder of the specimens were preserved in 70% ethanol and housed in the collection of the Monte L. Bean Life Science Museum at Brigham Young University. Laboratory Procedures Total genomic DNA was extracted from the frozen tissues using a standard proteinase K extraction followed by the addition of phenol/chloroform and precipitation with isopropanol. DNA was then dried and resuspended in TE buffer. PCR products were amplified using the following primersITS1: GTAAAAGTCGTAACAAGG and TCCTCCGCTWAWTGATATGC; ITS2: TGYGAACTGCAGGACACA and TGTGTCCTGCAGTTCRCA (5939). Standard PCR reactions were carried out on a Perkin-Elmer 9600 machine with 35 cycles and an annealing temperature of 508C. Fresh PCR products were cloned using the TOPO TA cloning kit (Invitrogen). Colonies containing the vector including the cloned PCR product were picked with a sterile pipette tip and put into 20 ml of water. This was shaken for 20 min, and then 1 ml was taken as the DNA template for an additional PCR, following the specifications suggested by the cloning kit literature (25 cycles with a 558C annealing temperature). Successful PCR products were purified using a GeneClean II kit (Bio 101). Automated sequences were generated on an ABI 377XL automated sequencer using the ABI Big-dye Ready-Reaction kit following the standard cycle sequencing protocol but using a quarter of the suggested reaction size. Phylogeny Reconstruction Sequences were aligned using CLUSTAL W (Thompson, Higgins, and Gibson 1994). Some adjustments were made by eye. The sequences were then imported into PAUP* (Swofford 1999) for phylogenetic analyses. When estimating phylogenetic relationships among sequences, one assumes a model of evolution Orconectes longidigitus . . . . . . . Orconectes luteus1 . . . . . . . . . . . O. luteus2 . . . . . . . . . . . . . . . . . . . O. luteus3 . . . . . . . . . . . . . . . . . . . O. luteus4 . . . . . . . . . . . . . . . . . . . Orconectes macrus . . . . . . . . . . . Orconectes neglectus . . . . . . . . . Orconectes punctimanus . . . . . . Orconectes virilis . . . . . . . . . . . . Procambarus clarkii1 . (...truncated)