Evidence for the Evolution of Bdelloid Rotifers Without Sexual Reproduction or Genetic David Mark Welch and Matthew Meselson

H. constricta was isolated from ground moss in Sandwich, MA, USA by M.M.; P. roseola was purchased from Carolina Biological Supply; and M. quadricornifera, A. vaga, and S. nebaliae were provided by Claudia Ricci and Giulio Melone. B. plicatilis strains AUS and RUS were provided by Terry Snell, B. calyciflorus was purchased from Florida Aquaculture, and E. ehrenbergi and S. socialis were provided by Robert Wallace. M. moniliformis DNA prepared from a single individual was provided by James Garey.

PCR, Cloning, and Sequencing. All PCR primers contained 5´ leaders and Xba I (sense primers) or Hind III (antisense primers) recognition sequences. Primers for the hsp82 region corresponding to D. melanogaster codons 13 to 302 (GenBank DROHSP82) (28) were ATCG TCTAGA GAR ACN TTY GCN TTY CAR GCN GA and ACGT AAGCTT RTG RTC YTC CCA RTC RTT NGT. Primers for the region corresponding to codons 151 to 321 were ACG TCTAGA Y GAY GAR CAR TAY GTN TGG GAR T and ACGT AAGCTT DAT RAA IAG NAG IGC NCG RAA YTC. Primers for the tbp region corresponding to D. melanogaster codons 187 to 295 (DROTATABF) were TGC TCTAGA CAR AAY ATH GTN WSI ACN GTN AAY and ACGT AAGCTT NGG RAA IAR YTC NGG YTC RTA. Primers for the rpol3I region corresponding to Saccharomyces cerevisiae codons 867 to 1098 (SCRPO31) were ACGT TCTAGA GAR TTY TTY TTY CAY GCN ATG GG and ACGT AAGCTT GT CAT YTG NGT NGC NGG YTC NCC. Primers for the tpi region corresponding to D. melanogaster codons 71 to 242 (DMTPIG) were ACG TCTAGA GGN GCN TTY ACN GGN GAR AT and ACGT AAGCTT AT RTC NAC RAA YTC NNG YTT.

Amplification reactions contained 100 to 500 ng of genomic DNA, 2 µg of each primer, 5 U of Taq polymerase, and 0.1 U of Pfu polymerase in 50 µl of 150 µM dNTPs, 10 mM tris (pH 9.0), 50 mM KCl, and 1% Triton X-100. Amplification was performed by 10 cycles of 1 min at 95°C; 1 min at 50° to 55°C; 1 min at 72°C, followed by 30 to 35 cycles of 30 s at 95°C; 45 s at 50° to 55°C; 1 min at 72°C. Reaction mixtures were separated on agarose gels and products of the expected size were excised, digested with Xba I and Hind III, cloned into pBSKII, and sequenced according to standard protocols.