208 
Fishery Bulletin 106(2) 
Table 2 
Mitochondrial DNA (mtDNA) 12S rRNA-control region (563 base pairs [bp]) fragment sizes resulting from the BsmAl and Hinll- 
recognizing sites during nucleotide sequence analyses of three Panulirus species: California spiny lobster ( Panulirus interrup- 
tus), blue spiny lobster (P. inflatus), and green spiny lobster (P. gracilis). Hap = haplotype nomenclature (see text for explanation); 
% = percentage of haplotypes in a sample of 215 sequences. Composite haplotypes were obtained by combining the haplotype of 
each enzyme. 
Species 
Enzyme 
Fragment size (bp) 
Hap 
% 
Composite haplotype 
P. interruptus 
BsmAl 
162,401 
A 
100.0 
AA 
Hindi 
440,123 
A 
100.0 
P. inflatus 
BsmAl 
162, 401 
A 
100.0 
AB 
Hindi 
563 
B 
100.0 
P. gracilis 
BsmAl 
115,47,401 
B 
100.0 
BA/BB 
Hinfl 
440,123 
A 
98.7 
563 
B 
1.3 
the amplified product delineated all three species in a 
1% agarose gel. The fragment size ofP. interruptus (with 
the use of LanCR-R primer) was -1000 bp, the fragment 
size of P. inflatus (with the use of PinRCl-b) was -800 
bp, and the fragment size of P. gracilis (with PgraRCl- 
b) was -700 bp (Fig. 4). Even though two P. interruptus 
specimens produced unspecific amplifications, the frag- 
ments stained weakly compared to the 1000-bp fragment 
and did not interfere with identification. Because the 
specimens analyzed by multiplex PCR were collected at 
99 
p Hap 4 
Hap 7 
Hap 5 
Hap 8 
■— Hap 6 
, 
r; 
99 
100 
0.02 
Genetic distance 
L 
different sites, these data show that there is no apparent 
intraspecific variation, which indicates that the multi- 
plex primer set provides a method that can be used to 
identify the three lobster species. 
Larval identification 
Species identification based on PCR-RFLP did not sup- 
port the identification based on morphological crite- 
ria. The fragment patterns produced by digestion with 
BsmAl and Hinfl provided a means for identi- 
fying P. inflatus and P. gracilis. Two specimens 
in each of groups 1 and 2 were identified as P. 
gracilis. The 42 remaining larvae belonging 
to groups 1, 2, 3, and 4 were identified as P. 
inflatus; none of the specimens were identi- 
fied as P. interruptus. Multiplex PCR analysis 
confirmed the results. 
- Hap 10 
Hap 9 
- Hap 1 1 
r Hap 2 
Hap 1 
Hap 3 
B 
Figure 2 
Neighbor-joining phylogenetic tree based on the mitochondrial 16S 
rRNA gene haplotypes (Hap 1-11) from a total of 20 adult lob- 
sters of blue spiny lobster (Panulirus inflatus), green spiny lobster 
(P. gracilis), and California spiny lobster (P. interruptus). Haplo- 
type groups identified as A, B, and C represent blue, green, and 
California spiny lobster, respectively. Tree reconstruction was based 
on Kimura’s two-parameter distance (K2P) with 1000 replications. 
Numbers on the nodes are bootstrap values. The branch length is 
measured as the number of nucleotide substitutions. 
Discussion 
Several methods with molecular markers have 
been carried out for identification at the species 
level. Even though PCR-RFLP and multiplex- 
PCR are widely used as tools in distinguishing 
species of different taxonomic groups (Moore 
et al., 2003; Chow et al., 2006a), efforts have 
to be made to obtain the proper amplification 
primers when universal primers do not give 
consistent results in the studied species. 
According to our analysis, identification 
of spiny lobster species can be successfully 
done by combining different strategies. First, 
although insufficient for recognizing spiny 
lobsters larvae, morphological criteria should 
be used. Then, one or both of the genetic tech- 
niques can be applied to definitively support 
the morphological results. The simultaneous 
use of the PCR-RFLP and multiplex PCR 
