210 
Fishery Bulletin 106(2) 
Panulirus interruptus Panulirus inflatus Panulirus gracilis 
12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 
Figure 4 
Polymerase chain reaction (PCR) products with the multiplex-PCR 
method for each of the three lobster species: California spiny lob- 
ster ( Panulirus interruptus), blue spiny lobster ( P . inflatus), and 
green spiny lobster {P. gracilis). Lanes 8, 15, and 21 are 100-bp 
ladder molecular markers. 
techniques is recommended because there are instances 
for which unsuccessful amplifications are obtained by 
one of the methods. This is a consequence of mutations 
in the annealing sites, rather than a failure of PCR 
techniques (Ray et al., 2002). 
The 20 sequences that we evaluated for differences 
in restriction patterns revealed species-specific restric- 
tion sites for BsmAl and Hinfl. Amplified products of 
previously identified adult lobsters were concordant 
with the restriction patterns found in these sequences. 
The PCR-RFLP technique is successful for separating 
Panulirus larvae because 16S rRNA is relatively easy 
to amplify at intraspecific conservative sites and inter- 
specific variable sites. 
The PCR-RFLP has also been applied to the cy- 
tochrome oxidase I (COI) gene to identify 10 spiny 
lobster species of the genus Panulirus found in the 
northwestern Pacific Ocean (Chow et al., 2006a). In 
general, Chow et al. (2006a) found more than two hap- 
lotypes per species; however, no composite haplotype 
was shared by these species. Although Chow et al. 
(2006a) suggested increasing efforts in searching for 
intraspecific variation on larger samples, the probabil- 
ity of misidentification is very low because substantial 
divergence has been observed among species of the 
genus Panulirus (Ptacek et al., 2001). Our results sup- 
port the finding of high divergence among the three 
lobster species in Mexico on the basis of nucleotide 
intraspecific variation (Fig. 2). 
Multiplex-PCR fragments are clearly capable of dis- 
criminating among the three lobster species. This meth- 
od is fast, simple, and relatively inexpensive because 
species identification can be performed by using just 
PCR amplifications with no digestion and with small 
reaction volumes. The multiplex-PCR method used in 
this study is successful because it is based on spe- 
cies-specific primers and a sufficiently high anneal- 
ing temperature (Tm) of 59°C is used that avoids the 
amplification of unspecific PCR products. Also, a high 
Tm reduces the possibility of amplifying homologous 
sequences from other species. 
Identification of lobster larvae is consistent with 
the restriction patterns found in adult lobsters and 
identifications carried out by PCR-RFLP can be cor- 
rectly confirmed by using multiplex PCR. Molecular 
analyses showed that the previous larval classifica- 
tion (based on morphological characters) was probably 
incorrect because very small or injured specimens 
were used. Alternatively, misidentification in lob- 
ster larvae could also be a consequence of morpho- 
logical criteria that are not diagnostic characters 
for discriminating between lobster larvae, as it has 
been reported in other Panulirus species, such as the 
Japanese spiny lobster (P. japonicus) and species from 
the Atlantic Ocean (i.e. , the Caribbean spiny lobster, 
P. argus) (Silberman and Walsh, 1992; Chow et al., 
2006a, 2006b). Morphological criteria currently used 
for the identification of phyllosoma larvae of P. inter- 
ruptus, P. inflatus, and P. gracilis are insufficient as 
well. Further descriptions of larvae specimens should 
be conducted to search for consistent morphological 
differences between species. 
Other possible applications of PCR-RFLPs and mul- 
tiplex PCR are used in fishery forensics, when lobster 
