186 
Fishery Bulletin 106(2) 
mykiss OMU34341, Z18683), tub gurnard ( Trigla lucerna 
Z18768), Atlantic mackerel ( Scomber scombrus Z18693), 
zebrafish ( Brachydanio rerio AJ306603), Pimelodella 
cristata (AJ306596), Atlantic herring ( Clupea harengus 
Z18764), dab ( Limanda limanda Z18681), brown bullhead 
( Ictalurus nebulosus Z18678), spotted green pufferfish 
( Tetraodon nigroviridis AJ270038, AJ270037), common 
carp (Cyprinus carpio AF133089), black ghost ( Apterono - 
tus albifrons AJ306595), Eurasian minnow ( Phoxinus 
phoxinus AJ306604), Synodontis clarias (AJ306597), 
Orinocodoras eigenmanni (AJ306606), angler ( Lophius 
piscatorius Z18765), European pilchard {Sardina pilchar- 
dus Z18767), coelacanth ( Latimeria chalumnae U34336), 
ghost shark ( Callorhinchus milii AY049812), Florida gar 
( Lepisosteus platyrhynchus Z18680), arawana ( Osteo - 
glossum sp. Z18684), rabbit fish ( Chimaera monstrosa 
Z18674), European eel ( Anguilla anguilla Z18673), 
bowfin ( Amia calva Z18672), and browneye skate ( Raja 
schmidti AF278683). 
DNA purification 
For adult specimens, approximately 50-100 mg of muscle 
tissue or a clipping from the fin was collected from 
either frozen or ethanol-preserved specimens and DNA 
was extracted with a Roche High Pure PCR Template 
Preparation Kit (Roche Diagnostics GmbH, Mannheim, 
Germany) following the manufacturer’s protocol for 
isolation of nucleic acids from mammalian tissue. The 
one exception to the manufacturer’s protocol was that 
we used a volume of 50 pL to elute the DNA in the final 
step of the procedure. For ethanol-preserved specimens, 
the tissues were rehydrated in three changes of sterile 
tris ethylenediaminetetraacetic acid (TE) buffer for 30 
minutes each. Tissues were then transferred to the 
cell lysis solution, and DNA was purified following the 
manufacture’s protocol. 
DNA extraction from larval fish was accomplished 
by first placing them in separate sterile 55 mm Pe- 
tri dishes and allowing them to rehydrate in sterile 
TE buffer [lOmM Tris HC1 (pH 7.4), ImM disodium 
ethylenediaminetetraacetate (EDTA) (pH 8.0)] for 30 
minutes. Next, an eye was microdissected with fine 
forceps and the DNA was purified as described above. 
This limited the physical damage to the larvae, allow- 
ing them to be used for future meristic studies. The 
forceps used for microdissection were decontaminated 
between specimens as described above. 
PCR amplification, cloning, and sequencing procedures 
for ITS regions of adult fish 
The PCR amplification reaction mixture used to amplify 
adult fish ITS regions is listed in Table 2 . DNA was PCR 
amplified in a MJ Research MiniCycler (MJ Research, 
Waltham MA) under the following touchdown cycling 
conditions: 2 min. at 95°C, 35 cycles each consisting of 
40 sec. denaturation at 95°C, 40 sec. initial annealing 
temperature at 64°C which decreased by 0.5°C per cycle 
for six cycles and 61°C thereafter and an extension of 
1.5 min. at 72°C. This procedure was followed by a 
final extension of 5 min. at 72°C. A 4-pL aliquot of each 
PCR reaction was checked for the presence of a specific 
amplification product by agarose gel electrophoresis 
(2% agarose, tris acetate EDTA fTAE gel], 50 volts) and 
ethidium bromide staining. PCR reactions containing 
specific products were cloned into the plasmid vector 
pCR2.1 by using the Topo TA Cloning Kit and by follow- 
ing the manufacturer’s protocol (Invitrogen, Carlsbad, 
CA). Plasmids were isolated and purified with a QIAprep 
Spin Miniprep Kit (Qiagen, Valencia, CA) and sequenced 
with an ABI377 DNA sequencer employing the Deoxy 
Terminator Cycle sequencing kit (Applied Biosystems- 
ABI, Foster City, CA). In addition to the ten local reef 
fishes, three adult fish of each Centropristis species 
were PCR amplified and cloned. DNA templates were 
sequenced completely in both directions with the M13F, 
M13R, Fish5.8SF, Fish5.8SF2, and Fish5.8SR prim- 
ers (Fig. 2A, Table 1). The resulting 3' SSU to 5' LSU 
sequence for each species was assembled by using the 
Vector NTI program (Informax Inc., 
Bethesda, MD) and submitted to 
GenBank (EF472464-EF472500). 
Phylogenetic analysis 
labile 1 
Primers used in this study to amplify and sequence the 3' end of the small sub- 
unit gene, internal transcribed spacer 1, 5.8S gene, internal transcribed spacer 
2, and the first approximately 48-bp of the large subunit gene 
Primer name 
Sequence (5'— 3') 
Forward sequencing 
M13F (plasmid vector) 
NSF1787F (universal forward) 
Fish5.8SF 
Fish5.8SF2 
GTAAAACGACGGCCAG 
CCGTAGGTGAACCTGCGG 
AGCTGCGAGAACTAATGTGAA 
TGCTCTGCTCGGGCTGTAGCG 
Reverse sequencing 
M13R (plasmid vector) 
FishLSU5’Rev (universal reverse) 
Fish5.8SR 
CAGGAAACAGCTATGAC 
CTTAAATTCAGCGGGTTGTCT 
TTCACATTAGTTCTCGCAGCT 
The ITS rDNA gene sequence from 
C. striata, C. ocyurus, C. philadel- 
phica , and Diplectrum formosum (for 
an outgroup) were aligned using the 
CLUSTAL-X algorithm (Thompson 
et ah, 1997). A Bayesian phylogenetic 
analysis of the aligned sequences 
was performed with the MrBayes 3.1 
program (Huelsenbeck et ah, 2001). 
Posterior probabilities were calcu- 
lated by using a Metropolis-coupled 
Markovian Chain Monte Carlo 
approach and with sampling con- 
ducted according to the Metropolis- 
