Amplification and Sequencing. To explore relationships within this family, we targeted an ≈1,000-bp region spanning the 3' end of the large ribosomal RNA (rrnL) gene, two leucine tRNAs, and a 5' region of the subunit 1 of the NADH dehydrogenase complex (nad1) gene. This region was amplified in two overlapping pieces: an ≈550-bp region of rrnL, using a standard primer pair 16SAR-L and 16SBR-H, and an overlapping ≈450-bp fragment spanning the 3' end of rrnL, L_CUN, and L_UUR, and the 5' end of nad1, using a newly designed primer pair 16SF (5'-TTGYGACCTCGATGTTGGA-3') and ND1R (WGCATCHGCAATAGGYTG). For each amplification 10-100 ng of each genomic DNA extraction was used as template in a 50-m l polymerase chain reaction (PCR) mixture that consisted of 1.5 mM MgCl₂, buffer supplied by the manufacturer (Promega: 50 mM KCl/10 mM Tris•HCl, pH 9.0 at 25°C/0.1% Triton X-100); 0.5 m M each primer; 200 m M each dNTP; and 1 unit of Taq DNA polymerase (Promega). PCRs were typically performed by using a Stratagene Robocycler. After 5 min of denaturation at 94° C and 5 min at 4° C, 1 unit of Taq DNA polymerase was added per reaction, and PCR was performed under the following cycling parameters: 94° C for 32 s, 50° C for 84 s, and 72° C for 90 s, for 38 cycles. Amplified products were extracted with chloroform, and a 5-m l aliquot of each PCR product was run out on a 1.4% agarose gel. When single bands of the appropriate size resulted from these amplifications, the PCR product was prepared for sequencing by using Geneclean III (Bio101). When supernumerary bands were present, the entire PCR product was run out on a 3% Nu-Sieve TAE agarose gel (BioWhittaker), and the band of the correct size was excised under long-wavelength UV and purified for sequencing by following the protocols provided with Geneclean III.