Watson et al.: Early larvae of Sebastes ensifer identified by molecular methods 
137 
5' CGA ACA GGA ART ATC AYT CTG G (Hyde and 
Vetter, 2009). In contrast to the protocols used for the 
CalCOFI specimens, this revised procedure provided 
a more robust amplification and saved both time and 
expense. Additionally, the use of only an eyeball, when 
possible, left the specimen otherwise intact for use in 
other studies. 
Haplotype sequences from both the CalCOFI and 
CCA sample sets were compared with reference data 
of 374 independent haplotype sequences representing 
67 Sebastes species (Hyde and Vetter, 2007) by using 
the software program PAUP*, vers. 4.0bl0 (Sinauer 
Associates, Sunderland, MA; Swofford, 2000) with the 
optimality criterion set to distance (number of base- 
pair fbp] differences divided by total number of bp se- 
quenced). Nonparametric bootstrapping was used (100 
replications, MAXTREES set to 1000) to cluster each 
larval haplotype within a database of consensus haplo- 
types (i.e., most common haplotype from a database of 
known adults) for putative identification. 
If the haplotype of a larva clustered with the single 
haplotype of a reference species with a bootstrap value 
>90%, the larva was identified as that species. Dis- 
tances between reference haplotypes and the unknown 
were examined to confirm that the unknown fell within 
the intraspecific diversity as a secondary confirmation 
of identification. If a larva clustered with a single ref- 
erence haplotype but the bootstrap value was <90%, 
a secondary analysis was performed that included all 
available haplotypes of at least the 3 nearest (in dis- 
tance) species to the larval haplotype; the haplotype 
was identified by comparison with those of the refer- 
ence species. Haplotype diversity for the reference spe- 
cies has a mean genetic distance of 0.0026 (correspond- 
